Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV.

Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed.     

Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse

We used the neonatal mouse model of rotavirus infection to study extraintestinal spread following oral inoculation. Five-day-old pups were inoculated with either SA11-Cl3, SA11-Cl4, SA11-4F, RRV, or B223. By using virus detection in the liver as a proxy determination for extraintestinal spread, rotavirus strains capable of extraintestinal spread at high frequency (rhesus rotavirus [RRV]) and very low frequency (SA11-Cl4) were identified. Both strains productively infected the gastrointestinal tract. Oral inoculation of mice with RRV/ SA11-Cl4 reassortants and determination of virus titers in the gut and liver revealed that the extraintestinal spread phenotype segregated with RRV genome segment 7 to a high level of significance (P = 10(-3)). RRV segment 7 also segregated with the growth of virus in the gut (P = 10(-5)). Although infection of the gut was clearly required for tropism to the liver, there was no correlation between virus titers in the gut and detection of virus in the liver. Five days after intraperitoneal administration to bypass the gut barrier to virus spread, RRV and SA11-Cl4 both were recovered in the liver. However, only RRV was found in the liver following subcutaneous inoculation, suggesting that this peripheral site presented a similar barrier to virus spread as the gut. Sequence analysis of segment 7 from parental RRV and SA11-Cl4 and selected reassortants showed that (i) amino acid differences were distributed throughout the coding sequences and not concentrated in any particular functional motif and (ii) parental sequence was preserved in reassortants. These data support the hypothesis that NSP3, coded for by genome segment 7, plays a significant role in viral growth in the gut and spread to peripheral sites. The mechanism of NSP3-mediated tropism is under investigation.     

A lymphatic mechanism of rotavirus extraintestinal spread in the neonatal mouse

We used the neonatal mouse model of rotavirus infection and virus strains SA11-clone 4 (SA11-Cl4) and Rhesus rotavirus (RRV) to examine the mechanism of the extraintestinal spread of viruses following oral inoculation. The spread-competent viruses, RRV and reassortant R7, demonstrated a temporal progression from the intestine, to the terminal ileum, to the mesenteric lymph nodes (MLN), and to the peripheral tissues. SA11-Cl4 was not found outside the intestine. Reassortant virus S7, which was unable to reach the liver in previous studies (E. C. Mossel and R. F. Ramig, J. Virol. 76:6502-6509, 2002), was recovered from 60% of the MLN, suggesting that there are multiple determinants for the spread of virus from the intestine to the MLN. Phenotypic segregation analysis identified RRV genome segment 6 (VP6) as a secondary determinant of the spread of virus to the MLN (P = 0.02) in reassortant viruses containing segment 7 from the spread-incompetent parent. These data suggest that in the orally infected neonatal mouse, the extraintestinal spread of rotavirus occurs via a lymphatic pathway, and the spread phenotype is primarily determined by NSP3 and can be modified by VP6.     

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.     

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.     

Genetic Analysis of Orbiviruses by Using RNase T(1) Oligonucleotide Fingerprints

Corresponding double-stranded RNA segments of the related orbiviruses Wallal and Mudjinbarry produced distinctly different RNase T₁ fingerprint patterns. No extensive sequence reiteration was observed between segments of Mudjinbarry virus. Fingerprint analysis of the genome of recombinant orbiviruses confirmed segment reassortment as a mechanism of interchange of genetic information. When temperature-sensitive mutants of each virus were crossed in mixed infection, a consistent pattern of segment reassortment was correlated with generation of the wild-type phenotype. Thus, the temperature-sensitive lesion of group II Wallal serogroup mutants was mapped to segment 1. The group I mutant lesion appears to be located in segment 2.     

Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.     

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3' termini ended with 5'-UGAACC-3' instead of the 3' consensus sequence 5'-UGACC-3', which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3' consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3' consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3' end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3' consensus sequence.     

Rotavirus RNA replication: VP2, but not VP6, is necessary for viral replicase activity.

Temperature-sensitive mutants of simian rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups on the basis of genome reassortment studies. Two of these mutants, tsF and tsG, map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. To gain insight into the role of these proteins in genome replication, MA104 cells were infected with tsF or tsG and then maintained at permissive temperature (31 degrees C) until 9 h postinfection, when some cells were shifted to nonpermissive temperature (39 degrees C). Subviral particles (SVPs) were recovered from the infected cells at 10.5 and 12 h postinfection and assayed for associated replicase activity in a cell-free system shown previously to support rotavirus genome replication in vitro. The results showed that the level of replicase activity associated with tsF SVPs from cells shifted to nonpermissive temperature was ca. 20-fold less than that associated with tsF SVPs from cells maintained at permissive temperature. In contrast, the level of replicase activity associated with tsG SVPs from cells maintained at nonpermissive temperature was only slightly less (twofold or less) than that associated with tsG SVPs from cells maintained at permissive temperature. Analysis of the structure of replicase particles from tsG-infected cells shifted to nonpermissive temperature showed that they were similar in size and density to virion-derived core particles and contained the major core protein VP2 but lacked the major inner shell protein VP6. Taken together, these data indicate that VP2, but not VP6, is an essential component of enzymatically active replicase particles.     

Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.     

Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein

Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines.     

Comparative molecular characterization of gene segment 11-derived NSP6 from lamb rotavirus LLR strain used as a human vaccine in China

Sequence-length polymorphism is known for rotavirus genetic segment 11 (encodes non-structural protein, NSP6). With the exception of 11 strains that have the coding potential for a 98-residue NSP6, majority of the strains have the potential for a 92-residue NSP6. In nine strains, the coding potential for this protein is even shorter. This report focuses on the NSP6 gene nucleotide sequence of Lanzhou Lamb Rotavirus (LLR) strain and its comparative molecular characterization. The LLR strain is a G10 P12 type, which is in use as a licensed human vaccine in China. The LLR NSP6 was compared with 56 other rotaviral NSP6 sequences including a rhesus strain (RRV) available in the database. Analyses indicate that while RRV-NSP6 belongs to the majority (92-residue) group, the LLR NSP6 belongs to the 98-residue group. When the rotavirus NSP6 protein was expressed in cells as GFP fusion protein from human, simian and the LLR strains, they all demonstrated punctate cytoplasmic distribution and, contrary to the computer-aided prediction, the NSP6 did not undergo phosphorylation, which in itself is a novel observation for the rotavirus NSP6.     

Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus.

Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.     

Physical mapping of DNA-temperature-sensitive mutations of vaccinia virus

Four DNA-temperature-sensitive (ts) mutations were mapped in the genome of vaccinia virus (VV). Physical mapping of these mutations was performed by restriction analysis of the genomes of recombinants between VV DNA- ts mutants and ectromelia virus as well as by the marker rescue with cloned restriction fragments of VV DNA. One of the mutations was mapped on the HindIII-E-fragment. Biochemical studies of this mutant indicate that the mutation is not in the DNA polymerase gene which is located on the same fragment. The other three mutations were mapped in a 10 kilobase region in the middle of the HindIII-D-fragment. As shown previously, these mutations inactivate different genes, and the products of these genes participate directly in the DNA synthesis. Thus, at least three proteins involved in the VV DNA synthesis are encoded by neighboring genes in the central part of the viral genome.     

Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.     

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.     

Comparison of the genomes of simian, bovine, and human rotaviruses by gel electrophoresis and detection of genomic variation among bovine isolates.

By co-electrophoresis in polyacrylamide gels, the segmented double-standed RNA genome of the simian rotavirus, SA 11, was compared with those of human and bovine rotaviruses. A comparison between SA 11 virus and the Northern Ireland cell culture adapted bovine virus showed that the electrophoretic mobilities of each of the 11 corresponding segments differed. In other comparisons, four to seven segment variations were more common. When the genomes of various bovine rotaviruses were compared, eight different electropherotypes were detected. Four of these electropherotypes were obtained from one property during a single outbreak of disease. In view of such genetic diversity, a scheme for the systematic designation of different rotavirus samples is proposed. The significance of the variations in relation to the molecular epidemiology of bovine rotavirus infections is discussed.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination.

Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome.     

Genetic and phenotypic analysis of herpes simplex virus type 1 mutants conditionally resistant to immune cytolysis

Nine temperature-sensitive (ts) mutants of herpes simplex virus type 1 selected for their inability to render cells susceptible to immune cytolysis after infection at the nonpermissive temperature have been characterized genetically and phenotypically. The mutations in four mutants were mapped physically by marker rescue and assigned to functional groups by complementation analysis. In an effort to determine the molecular basis for cytolysis resistance, cells infected with each of the nine mutants were monitored for the synthesis of viral glycoprotein in total cell extracts and for the presence of these glycoproteins in plasma membranes. The four mutants whose ts mutations were mapped were selected with polypeptide-specific antiserum to glycoproteins gA and gB; however, three of the four mutations mapped to DNA sequences outside the limits of the structural gene specifying these glycoproteins. Combined complementation and phenotypic analysis indicates that the fourth mutation also lies elsewhere. The ts mutations in five additional cytolysis-resistant mutants could not be rescued with single cloned DNA fragments representing the entire herpes simplex virus type 1 genome, suggesting that these mutants may possess multiple mutations. Complementation tests with the four mutants whose ts lesions had been mapped physically demonstrated that each represents a new viral gene. Examination of mutant-infected cells at the nonpermissive temperature for the presence of viral glycoproteins in total cell extracts and in membranes at the cell surface demonstrated that (i) none of the five major viral glycoproteins was detected in extracts of cells infected with one mutant, suggesting that this mutant is defective in a very early function; (ii) cells infected with six of the nine mutants exhibited greatly reduced levels of all the major viral glycoproteins at the infected cell surface, indicating that these mutants possess defects in the synthesis or processing of viral glycoproteins; and (iii) in cells infected with one mutant, all viral glycoproteins were precipitable at the surface of the infected cell, despite the resistance of these cells to cytolysis. This mutant is most likely mutated in a gene affecting a late stage in glycoprotein processing, leading to altered presentation of glycoproteins at the plasma membrane. The finding that the synthesis of both gB and gC was affected coordinately in cells infected with six of the nine mutants suggests that synthesis of these two glycoproteins, their transport to the cell surface, or their insertion into plasma membranes is coordinately regulated.