RESUMO
During last few decades, role of microbiota and its importance in several diseases has been a hot topic for research. The microbiota is considered as an accessory organ for maintaining normal physiology of an individual. These microbiota organisms which normally colonize several epithelial surfaces are known to secrete several small molecules leading to local and systemic effects on normal biological processes. The role of microbiota is also established in carcinogenesis as per several recent findings. The effects of microbiota on cancer is not only limited to their contribution in oncogenesis, but the overall susceptibility for oncogenesis and its subsequent progression, development of coinfections, and response to anticancer therapy is also found to be affected by microbiota. The information about microbiota and subsequent contributions of microbes in anticancer response motivated researchers in development of microbes-based anticancer therapeutics. We provided current status of microbiota contribution in oncogenesis with special reference to their mechanistic implications in different aspects of oncogenesis. In addition, the mechanistic implications of bacteria in anticancer therapy are also discussed. We conclude that several mechanisms of microbiota-mediated regulation of oncogenesis is known, but approaches must be focused on understanding contribution of microbiota as a community rather than single organisms-mediated effects.
Assuntos
Microbiota , Neoplasias/etiologia , Humanos , Neoplasias/microbiologiaRESUMO
The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.
Assuntos
Endométrio/fisiologia , Proteínas da Gravidez/fisiologia , Prenhez/fisiologia , Proteômica , Ovinos/fisiologia , Animais , Eletroforese em Gel Bidimensional , Desenvolvimento Embrionário/fisiologia , Estro/fisiologia , Feminino , Espectrometria de Massas , Gravidez , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. METHODS: SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. RESULTS: Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. CONCLUSION: Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.
Assuntos
Anti-Inflamatórios/química , Peptídeos/química , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Oncostatina M , Inibidores da Tripsina/farmacologiaRESUMO
The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins.
Assuntos
Doenças Cardiovasculares/diagnóstico , Neoplasias/diagnóstico , Doenças do Sistema Nervoso/diagnóstico , Proteoma , Biomarcadores , Biomarcadores Tumorais , Biologia Computacional , Eletroforese em Gel Bidimensional , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Análise de Sequência de ProteínaRESUMO
Enteroviruses have been implicated in persistent infections of the nervous system and in certain paralytic motor neuron syndromes. Enteroviral persistence may depend on defective transcription, resulting in the abnormal production of equal amounts of genomic and template RNA strands rather than the normal ratio of 60-100:1. An in vitro model of a persistent coxsackie virus in human skeletal muscle cells was investigated using in situ hybridisation and a semiquantitative parallel, complementary, reverse transcriptase polymerase chain reaction. The ratio of genomic to template RNA was found to be approximately 60:1. We conclude that enteroviral persistence in this in vitro model is not dependent on altered transcription. In vivo, other viral and host factors should be considered.
Assuntos
Enterovirus Humano B/genética , Regulação Viral da Expressão Gênica , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Transcrição Gênica , Enterovirus Humano B/fisiologia , Genoma Viral , Humanos , Hibridização In Situ , Músculo Esquelético/virologia , Reação em Cadeia da Polimerase , Rabdomiossarcoma/patologia , Moldes Genéticos , Células Tumorais Cultivadas , Latência Viral , Replicação ViralRESUMO
An 18-residue-long fragment of vasoactive intestinal polypeptide [VIP(11-28)-NH2] that is known to be analgesic was synthesized by solid-phase t-Boc methodology on a 4-methylbenzhydrylamine resin. Circular dichroism spectroscopy gave evidence that the peptid acquires about 60% helical structure in 50/50 methanol/phosphate buffer, pH 6.0, and 65% (+/-5%) helicity in 80/20 methanol/phosphate buffer pH 7.0, A 2.0 mM solution of VIP (11-28) NH2 in 80% methanol, 20% phosphate buffer pH 7.0 was subjected to 2-dimensional nuclear magnetic resonance (NMR) studies The NMR results suggested formation of an extended helical structure extending from residue 11 to 27 essentially the same region found to be helical in a VIP(1-28)-NH2 and log. This finding suggests that the sequence required for analgesia assumes a helical structure at the receptor.
Assuntos
Analgésicos/química , Fragmentos de Peptídeos/química , Peptídeo Intestinal Vasoativo/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Estrutura Secundária de Proteína , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
Attempts to evaluate the relative levels of enteroviral genomic and template RNA strands in small biopsy tissue samples from patients have yielded ambiguous data, largely due to the limited amount of RNA available. A novel semi-nested polymerase chain reaction (PCR) technique was developed to enable RNA levels to be examined more accurately. PCR products were visualised by horizontal agarose gel electrophoresis. This technique was demonstrated on linear single-stranded plasmid DNA; viral RNA isolated from a human rhabdomyosarcoma (RD) cell line persistently infected with a mutated coxsackie B5 virus (piRD) and two cell lines, RD and HEp2 cells, acutely infected with a wild-type clinical isolate of coxsackie B5 virus.
Assuntos
Eletroforese em Gel de Ágar , Enterovirus Humano B/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Sequência de Bases , Humanos , Dados de Sequência Molecular , RNA Viral/química , DNA Polimerase Dirigida por RNA , Rabdomiossarcoma/virologia , Células Tumorais CultivadasRESUMO
Serotype 5 coxsackie B virus (CBV5) can establish in vitro persistent infections in human rhabdomyosarcoma (RD) cells. This paper describes the characterisation of the virus released from the persistently infected RD cell line designated piRD-3673. Although infectious virus was released for 42 sequential passages of piRD-3673 cells, no gross virus-specific cytopathic effect was detected when the cells were examined by light microscopy. Two-dimensional polyacrylamide gel electrophoresis was used to compare the virus released from piRD-3673 cells with the CBV5 isolate (CBV-3673) used to initiate the persistent virus infection. Two of the virus intracellular proteins (apparent molecular weights 33,000 and 39,000, designated p33 and p39, respectively) increased in their net basic charge for the virus released from piRD-3673 cells compared to CBV-3673; a reduction in the apparent molecular weights of p33 and p39 was also observed. The charge alteration for both p33 and p39 was a two-stage process, the accumulative effect of which resulted in p33 increasing in pI from 6.14 to 6.53 and p39 increasing in pI from 6.29 to 6.63. The first mutation of p33 and p39 occurred between passages 7 and 10 of piRD-3673 cells and affected both the charge and apparent molecular weight of these two proteins. The second mutation at passage 15 of piRD-3673 cells caused only a change in the charge of p33 and p39. Two other virus proteins (p54 and p75) showed no evidence of mutation over the same passage history of piRD-3673 cells. The virus released from piRD-3673 cells also differed from CBV-3673 by two further criteria, a reduction in plaque-forming efficiency in HEp-2 cells and increased virus replication in RD cells. These data on virus evolution are discussed in relation to the maintenance of persistent CBV infections and the presence of naturally occurring CBV variants.
Assuntos
Infecções por Coxsackievirus/genética , Eletroforese em Gel Bidimensional , Enterovirus Humano B/genética , Mutação , Proteínas Virais/genética , Enterovirus Humano B/crescimento & desenvolvimento , Enterovirus Humano B/fisiologia , Humanos , Ponto Isoelétrico , Peso Molecular , Rabdomiossarcoma/microbiologia , Células Tumorais Cultivadas , Proteínas Virais/químicaRESUMO
Infection of rhabdomyosarcoma (RD) cells by coxsackie B5 virus (CBV5) was non-cytopathic, although low titres of infectious virus were produced after 24 h post-infection. The extent of CBV5 replication in RD cells increased after sequential passage of the virus in these cells. The RD cells from the first cycle of CBV5 infection were recovered and maintained in culture for 3 months (equivalent to 21 passages) releasing infectious virus throughout this period; these cells were considered to be persistently infected with CBV5 and were designated piRD cells. Coxsackie virus antigen was demonstrated in a small proportion of piRD cells by immunofluorescence staining. High resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the intracellular proteins prepared from piRD cells, three proteins were detected which were absent in uninfected RD cells. These new proteins were similar in charge to virus proteins induced during CBV5 lytic infection of HEp-2 cells. Quantitative densitometry of 2-dimensional protein profiles of piRD and uninfected cells showed no significant disruption of RD cell protein synthesis by the persistent virus infection. Three cloned cell lines were recovered from piRD cells, none of which showed evidence of infectious virus or virus-induced protein synthesis suggesting that the parental cell line was a carrier culture for CBV5.
Assuntos
Enterovirus Humano B/fisiologia , Rabdomiossarcoma/microbiologia , Antígenos Virais/análise , Enterovirus Humano B/imunologia , Imunofluorescência , Células Tumorais Cultivadas/microbiologia , Replicação ViralRESUMO
Subtilisin, a bacterial serine protease, is secreted as pre-pro-subtilisin. Previously, we demonstrated that the pro-peptide moiety of intact pro-subtilisin can guide the folding of inactive protein to active enzyme both in an intramolecular (6) and intermolecular manner (18). Herein is reported the total chemical synthesis of the pro-sequence (77 amino acids) of pre-pro-subtilisin BPN' carried out by solid phase methods. The structure was confirmed by both sequencing and amino acid analysis of the fragment peptides resulting from a V-8 protease digest. Preliminary studies indicate that the synthetic pro-peptide itself can renature denatured subtilisin BPN'. This study demonstrates a novel method for examining protein folding with the aid of exogenously added synthetic peptides.
Assuntos
Precursores Enzimáticos/síntese química , Fragmentos de Peptídeos/síntese química , Peptídeos/síntese química , Subtilisinas/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Brometo de Cianogênio , Precursores Enzimáticos/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Serina Endopeptidases , Subtilisinas/metabolismoRESUMO
Human sera containing respiratory syncytial (RS) virus-specific antibodies enhance RS virus infection of the U937 macrophage cell line. There was an increase in the number of cells expressing virus antigen when U937 cells were infected with RS virus in the presence of human serum compared to cells infected in the absence of human serum. Human sera enhanced virus yield, as measured by the cell-released infectious virus, by an average of 50-fold compared to virus infection in the absence of human serum. The comparison of the enhancing activities of paired acute and convalescent human sera showed that the titre of enhancing antibody increased in parallel with the titre of RS virus-specific antibody measured by complement fixation and virus neutralization. An RS virus-specific neutralizing monoclonal antibody directed to the virus F protein enhanced virus infection of U937 cells. A non-neutralizing monoclonal antibody directed to the virus nucleoprotein did not enhance virus infection. The possible role of enhancing antibodies in vivo is discussed.
Assuntos
Anticorpos Antivirais/imunologia , Macrófagos/microbiologia , Vírus Sinciciais Respiratórios/imunologia , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Meios de Cultura , Humanos , Soros Imunes/imunologia , Pessoa de Meia-Idade , Vírus Sinciciais Respiratórios/fisiologia , Replicação ViralRESUMO
Three hybridoma antibodies, prepared against the RSN-2 strain of human respiratory syncytial (RS) virus, have been used to identify antigenic variation between 41 isolates of RS virus collected from widely separated geographical regions over a period of 29 years. One antibody was directed against an antigenic site on the virus fusion protein, VP70. This site was shared by 21 virus isolates tested and its recognition by the antibody was sensitive to the presence of 2-mercaptoethanol. The remaining two antibodies used react against the virus phosphoprotein, VPP32. Two independent sites were recognized on VPP32 by these antibodies. One antibody reacted with all of the virus isolates screened while the second reacted with only 21 out of the 41 virus isolates. On the basis of the variable epitope, two antigenic types of human RS virus were identified. The distribution of each antigenic group among 28 RS virus isolates from the Grampian Region, north-east Scotland, collected between 1982 and 1984 was determined. The reactivity of these antibodies was examined using immunofluorescence staining and by immunoblotting; the latter technique also revealed that the electrophoretic mobility of VPP32 varied in parallel with the variable antigenic site.
Assuntos
Antígenos Virais/genética , Variação Genética , Vírus Sinciciais Respiratórios/imunologia , Animais , Anticorpos Monoclonais , Antígenos Virais/isolamento & purificação , Carcinoma de Células Escamosas , Linhagem Celular , Chlorocebus aethiops , Imunofluorescência , Humanos , Rim , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/isolamento & purificação , Proteínas Virais/isolamento & purificaçãoRESUMO
The replication of Dugbe (DUG) virus, a member of the Nairovirus genus of the Bunyaviridae, has been investigated. During the infection of BS-C-1 cells a virus-specific c.p.e. was initially observed followed by recovery of the cell monolayer but with continued production of infectious virus. Six DUG virus-induced polypeptides were identified with apparent molecular weights, determined by gel electrophoresis, of 92000 (p92), 82000 (p82), 77000 (p77), 52000 (p52), 48000 (p48) and 34000 (p34). The polypeptides p77 and p34 were detected in purified DUG virions but not in extracts of virus-infected cells pulse-labelled with [3H]leucine. Polypeptides p48 and p52 were found in both purified virus preparations and in extracts of infected cells. p82 and p92 were found only in lysates of infected cells. When two-dimensional gel electrophoresis was used to analyse infected cells, p48 was found to have a net positive charge.
Assuntos
Bunyaviridae/metabolismo , Biossíntese Peptídica , Animais , Bunyaviridae/crescimento & desenvolvimento , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Camundongos , Peptídeos/análiseRESUMO
Monoclonal antibodies to the RSN -2 isolate of human respiratory syncytial (RS) virus have been characterized with regard to their specificity for viral polypeptides and for different RS virus isolates. Five hybridoma antibodies recognized the phospho-protein VPP32 and the other recognized the matrix protein VPM27 . Evidence was obtained to support the view that VPP32 was associated with the nucleoprotein VPN41 . Three of the antibodies to VPP32 showed cytoplasmic immunofluorescent staining while the other two showed only surface staining of virus-infected cells. The immunoblot technique was used to determine the immunoreactivity of four of the hybridoma antibodies against human RS isolates other than RSN -2. One of these antibodies reacted exclusively with RSN -2 virus isolate whereas the others detected determinants shared by all human RS isolates tested. Extension of this approach may offer the possibility of typing RS isolates using monoclonal antibodies.
Assuntos
Antígenos Virais/análise , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais/imunologia , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Epitopos , Imunofluorescência , Fosfoproteínas/imunologia , Vírus Sinciciais Respiratórios/classificaçãoRESUMO
The effect of monensin, a monovalent ionophore, on La Crosse virus particle formation and polypeptide synthesis was examined. Monensin inhibited the release of virus particles (both infectious and non-infectious) from infected BHK-21 cells. Monensin had no detectable effect on the synthesis of polypeptides G1, G2, and N.
Assuntos
Bunyaviridae/efeitos dos fármacos , Vírus da Encefalite da Califórnia/efeitos dos fármacos , Furanos/farmacologia , Monensin/farmacologia , Proteínas Virais/biossíntese , Animais , Linhagem Celular , Cricetinae , Vírus da Encefalite da Califórnia/crescimento & desenvolvimento , Vírus da Encefalite da Califórnia/metabolismo , Tunicamicina/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Analyses of the viral ribonucleic acids and structural polypeptides of 17-22 of the 119 accepted or proposed members of the Bunyavirus genus of arboviruses (family Bunyaviridae), have shown that from the standpoint of their structural components these viruses are highly comparable to each other. The average molecular weights for the three viral RNA species (L, large, M, medium, S, small) of 17 bunyaviruses were 2.93 X 10(6) (L, range 2.7-3.1 X 10(6)), 2.0 X 10(6) (M, range 1.8-2.3 X 10(6)), and 0.435 X 10(6) (Sm range 0.28-0.50 X 10(6)). The average molecular weights of the three major virion polypeptides (glycoproteins G1 and G2, and nucleocapsid protein, N) of 22 bunyaviruses were 115 X 10(3) (G1, range 108-120 X 10(3)), 37 X 10(3) (G2, range 20-41 X 10(3)) and 22 X 10(3) (N, range 19-25 X 10(3)). These results indicate that the structural components of bunyaviruses are different from those reported for Phlebotomus fever, Uukuniemi, and Crimean-Congo hemorrhagic fever, and other members of the Bunyaviridae family that are not currently assigned to a genus.
Assuntos
Bunyaviridae/análise , Peptídeos/análise , RNA Viral/análise , Proteínas Virais/análise , Animais , Vírus Bunyamwera/análise , Vírus da Encefalite da Califórnia/análise , Camundongos , Orthobunyavirus/análise , Vírus Simbu/análiseAssuntos
Desoxiaçúcares/farmacologia , Desoxiglucose/farmacologia , Vírus da Encefalite da Califórnia/efeitos dos fármacos , Vírus da Encefalite/efeitos dos fármacos , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Tunicamicina/farmacologia , Proteínas Virais/biossíntese , Relação Dose-Resposta a Droga , Vírus da Encefalite da Califórnia/metabolismo , Biossíntese Peptídica , TemperaturaRESUMO
The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.
Assuntos
Arbovírus/análise , Vírus Bunyamwera/análise , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Viral/análise , Animais , Vírus Bunyamwera/metabolismo , Linhagem Celular , Sistema Livre de Células , Cricetinae , Genes Virais , Rim , Ácidos Nucleicos Heteroduplexes , Biossíntese Peptídica , RNA/análise , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/biossínteseRESUMO
The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive (ts) mutants have been analyzed. The results obtained agree with in vitro studies (Bouloy et al., C.R. Acad. Sci. Paris 280:213-215, 1975; M. Bouloy and C. Hannoun, Virology 69:258-264, 1976; M. Ranki and R. Pettersson, J. Virol. 16:1420-1425, 1975) which have shown that bunyaviruses are negative-stranded RNA viruses with a virion RNA-directed RNA polymerase. The in vivo transcription studies have demonstrated that in the presence of protein synthesis inhibitors (puromycin or cycloheximide) SSH virus can synthesize viral complementary RNA (primary transcription) throughout the infection cycle. The increased levels of viral complementary RNA obtained in the absence of protein synthesis inhibitors (secondary transcription) were not markedly reduced if cells were pretreated with actinomycin D (5 mug/ml), alpha-amanitin (25 mug/ml), or rifampin (100 mug/ml), although progeny virus yields were reduced by up to 80% in the actinomycin D- and rifampin-treated cells. The in vivo transcription capabilities of SSH group I ts mutants at temperatures which were nonpermissive (40 degrees C) for virus replication gave values comparable to those obtained at permissive temperatures (33 degrees C). The SSH group I mutants appear, therefore, to be RNA-positive mutant types. When compared with their transcription capabilities at 33 degrees C, the in vivo transcription abilities of four SSH group II ts mutants (and one double group I/II ts mutant) were found to be more impaired at 40 degrees C than those of the SSH group I ts mutants or wild-type SSH virus at 40 degrees C, although the viral complementary RNA synthetic capabilities of these group II (and group I/II) mutants at 40 degrees C were significantly higher than their primary transcription capabilities (as measured at 33 degrees C in the presence of puromycin or cycloheximide). It was concluded, therefore, that these SSH group II (and double group I/II) ts mutants have an intermediate RNA phenotype. Hybridization studies using (32)P-labeled individual L, M, and S viral RNA species of SSH virus have demonstrated the presence of viral complementary RNA to all three species in extracts of cells infected with SSH ts II-30 and incubated at 33 degrees C (primary and secondary transcription) or 40 degrees C, a nonpermissive temperature for its replication. The results of pulse-labeled in vivo protein analyses indicated that greater quantities of intracellular N protein (coded for by S RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978]) than G1 and G2 polypeptides (coded for by M RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-776, 1979]) were present in extracts of cells infected with wild-type SSH virus. In extracts of SSH group I, II, or I/II ts mutant-infected cells incubated at 33 degrees C, N and G1, and for the group II mutant-infected cells, G2, viral polypeptides were detected, whereas in extracts obtained from group I or II mutant virus-infected cells incubated at 40 degrees C, low levels of N and G1 polypeptides were evident.