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At least 19 recordsLinked to original sources
PubMed2002

Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR.

Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >or=95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.

Animals↗
PubMed1974

Control of simian virus 40 gene expression in adenovirus-simian virus 40 hybrid viruses. Synthesis of hybrid adenovirus 2-simian virus 40 RNA molecules in cells infected with a nondefective adenovirus 2-simian virus 40 hybrid virus.

The effect of interferon on simian virus 40 (SV40) and adenovirus 2 (Ad2) T antigen synthesis has been examined in cells infected with SV40, with Ad2, and with a nondefective Ad2-SV40 hybrid virus, Ad2(+)ND(4). The induction of SV40 T antigen by SV40 was highly sensitive to interferon, whereas the induction of Ad2 T-antigen by Ad2 was resistant. This difference in interferon sensitivity was also noted in cells simultaneously infected with both viruses. However, the induction of SV40 T antigen by Ad2(+)ND(4), which contains covalently linked SV40 and Ad2 DNAs, was as resistant to interferon as the induction of Ad2 T antigen. This change in the interferon sensitivity of SV40 T antigen synthesis suggests that the expression of at least this portion of the SV40 genetic information in Ad2(+)ND(4) is under Ad2 genetic control. When RNA extracted from Ad2(+)ND(4)-infected cells was examined by means of sequential hybridization with Ad2 DNA, elution, and rehybridization with SV40 DNA, 27% of the SV40-specific RNA was found to be linked to Ad2 RNA. No such linkage was detected in control mixtures of Ad2 and SV40 RNAs. The presence of Ad2 and SV40 nucleotide sequences in the same RNA molecule implies that, in Ad2(+)ND(4) infection, transcription is initiated in the DNA of one virus (Ad2 or SV40) and continues without interruption across the point of junction into the DNA of the other virus. Furthermore, the interferon resistance of Ad2(+)ND(4)-induced SV40 T antigen synthesis suggests that transcription of the genetic information for SV40 T antigen is initiated in a region of Ad2 DNA.

Adenoviridae↗
PubMed1986

The association between serological titers in infectious bovine rhinotracheitis virus, bovine virus diarrhea virus, parainfluenza-3 virus, respiratory syncytial virus and treatment for respiratory disease in Ontario feedlot calves.

A seroepidemiological study of the association between antibody titers to infectious bovine rhinotracheitis, parainfluenza-3, bovine virus diarrhea and bovine respiratory syncytial viruses, and treatment for bovine respiratory disease was conducted. A total of 322 calves from five different groups were bled on arrival, then one month later all cases (cattle treated for bovine respiratory disease) were rebled together with an equal number of controls (cattle not treated for any disease). Titers to these viruses varied significantly from group to group. Based on seroconversion, infectious bovine rhinotracheitis virus was active in 4.4%, bovine virus diarrhea virus in 24%, parainfluenza-3 virus in 69.5% and bovine respiratory syncytial virus in 71.3% of the cattle. Cattle with low titers to infectious bovine rhinotracheitis and/or bovine respiratory syncytial viruses on arrival, were at increased risk of subsequent treatment for bovine respiratory disease. Treated cattle also had significantly greater increases to parainfluenza-3 and/or bovine virus diarrhea viruses than control calves. Treatment rates varied considerably from group to group and were not strongly correlated with weight gain in the postarrival period.

Animals↗
PubMed1969

In vitro transformation by the adenovirus-simian virus 40 hybrid viruses. V. Virus-specific ribonucleic acid in cell lines transformed by the adenovirus 2-simian virus 40 and adenovirus 12-simian virus 40 transcapsidant hybrid viruses.

The ribonucleic acid-deoxyribonucleic acid hybridization technique was utilized to determine the presence of adenovirus (ad) and SV40 genetic information and to determine which ad genomes were present in clones of hamster cells transformed with the ad 2-SV40 and ad 12-SV40 transcapsidant hybrid virus populations. The results were correlated with the morphology of the transformed cells and colonies. It was found that cells transformed by either transcapsidant virus which had an SV40 morphology contained the ad 7 and SV40 genomes, whereas cells with a typical ad morphology contained only ad genetic information. Cells and colonies with morphological features of both ad- and SV40-transformed cells contained either the ad 2, or ad 12 genomes, depending on the transcapsidant used, together with the ad 7 and SV40 genomes. The results indicate the following: at least three different events occurred during transformation of hamster cells by the transcapsidant virus populations; the morphology of the resulting clones is determined by the viral genome(s) present; the linkage of the ad 7-SV40 genomes is confirmed since the ad 7- SV40 genomes were never found to be dissociated; the defective ad 7-SV40 genomes are capable of causing transformation; and the transcapsidant particle is probably composed of only ad 7 and SV40 genetic information.

Adenoviridae↗
PubMed1998

Evaluation of a single dilution ELISA system for detection of seroconversion to bovine viral diarrhea virus, bovine respiratory syncytial virus, parainfluenza-3 virus, and infectious bovine rhinotracheitis virus: comparison with testing by virus neutralization and hemagglutination inhibition.

A single-dilution quantitative enzyme-linked immunosorbent assay (ELISA) system, based on commercial ELISA kits, for the simultaneous detection of seroconversion to bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI3V), and infectious bovine rhinotracheitis virus (IBRV) was evaluated by testing acute and convalescent serum pairs from 564 cattle in 145 outbreaks of respiratory disease. Seroconversion to BVDV, BRSV, PI3V and IBRV was detected in 8.0%, 19.0%, 13.7%, and 7.4%, respectively, of serum pairs tested. Seroconversion was detected in 60.7% of herds and 34.6% of animals tested. Infection with 2 or more viruses was found in 46.6% of these herds and in 27.2% of these animals. The majority of BVDV infections (62%) were associated with other virus infections, suggesting that BVDV may potentiate infection with other agents rather than being a primary pathogen of the respiratory tract. The results were compared with those obtained by virus neutralization and hemagglutination inhibition testing, and the sensitivity, specificity, and overall correlation were calculated. Sensitivities of 92%, 95%, 100%, and 100% were obtained for BVDV, BRSV, PI3V, and IBRV, respectively. The corresponding specificity values were 89%, 92%, 86%, and 91%. The overall correlation for each virus was 90%, 93%, 90%, and 93%, respectively. These results demonstrate that this ELISA system may be used successfully to detect seroconversion in serum pairs, highlight the frequency of multiple viral infections in outbreaks of respiratory disease, and provide further evidence of an immunosuppressive role for BVDV infections.

Animals↗
PubMed1998

Inactivation of human immunodeficiency virus type 1, hepatitis A virus, respiratory syncytial virus, vaccinia virus, herpes simplex virus type 1, and poliovirus type 2 by hydrogen peroxide gas plasma sterilization.

BACKGROUND: Studies were conducted to determine the capability of a hydrogen peroxide gas plasma sterilization process to inactivate several types of viruses. Six test agents were used: HIV type 1, human hepatitis A virus, respiratory syncytial virus, vaccinia, herpes simplex virus type 1, and poliovirus type 2. METHODS: The test viruses were suspended in cell culture medium and dried on the bottom of sterile glass petri dishes. The inoculated dishes were processed in the hydrogen peroxide gas plasma system for half the normal sterilization cycle time. Four inoculated carriers for each virus were used in two separate half cycles. Infectivity of the test viruses and cytotoxicity to the indicator cell lines were assayed. RESULTS: The hydrogen peroxide gas plasma sterilization process produced inactivation of the six viral test agents under these experimental conditions. The reduction in viral titers ranged from 2.5 log10 to 5.5 log10, a 99.68% to 99.999% decrease. CONCLUSIONS: These results clearly demonstrate the virucidal effectiveness of the hydrogen peroxide gas plasma sterilization process against both lipid and nonlipid viruses.

Cells, Cultured↗
PubMed1994

Lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus.

Lelystad virus (LV) is an enveloped positive-stranded RNA virus, which causes abortions and respiratory disease in pigs. The complete nucleotide sequence of the genome of LV has been determined. This sequence is 15.1 kb in length and contains a poly(A) tail at the 3' end. Open reading frames that might encode the viral replicases (ORFs 1a and 1b), membrane-associated proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7) have been identified. Sequence comparisons have indicated that LV is distantly related to the coronaviruses and toroviruses and closely related to lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). A 3' nested set of six subgenomic RNAs is produced in LV-infected alveolar lung macrophages. These subgenomic RNAs contain a leader sequence, which is derived from the 5' end of the viral genome. Altogether, these data show that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-stranded RNA viruses, the Arteriviridae.

Animals↗
PubMed1969

In vitro transformation by adenovirus-simiam virus 40 hybrid viruses. IV. Properties of clones isolated from cell lines transformed by adenovirus 2-simiam virus 40 and adenovirus 12-simiam virus 40 transcapsidant hybird viruses.

Clones were isolated from hamster cells transformed by the adenovirus 2-SV40 and adenovirus 12-SV40 transcapsidant hybrid viruses. The clones were characterized with respect to their cytomorphology, virus and antigen content, and the histomorphology of tumors induced by transplantation of the clonal sublines to hamsters. Three different cellular and colonial morphologies were observed. Clones with an SV40 morphology gave rise to tumors predominantly with an SV40 histology, whereas clones with an adenovirus morphology produced typical adenovirus tumors upon transplantation of the transformed cells. Clones which had features of both SV40 and adenovirus transformed cells gave rise to "intermediate" and adenovirus tumors. The results indicate that multiple events occur during transformation and tumorigenesis by the transcapsidant virus populations and provide an explanation for the multiplicity of findings which have been reported with these virus populations.

Adenoviridae↗
PubMedDate not supplied

Hybrids between rubella virus and a latent virus of baby hamster kidney cell line BHK21: characterization of rubella virus and type 2 hybrid virus genomes and determination of their physical homology.

The biochemical nature of rubella virus and type 2 hybrid virus, which is a recombinant between rubella virus and a latent retrovirus of BHK21 cells, has been characterized. Type 2 hybrid virus carries DNA polymerase able to copy exogenous DNA. However, disrupted type 2 hybrid virions do not synthesize detectable amounts of DNA using the endogenous viral RNA or synthetic poly(rA)/oligo(dT) primed as a template. Thus, the type 2 hybrid virus DNA polymerase has no detectable reverse transcriptase activity. Rubella virus and type 2 hybrid virus RNA can serve as templates for avian myeloblastosis virus (AMV) reverse transcriptase, although they are inefficient. The addition of oligo(dT) to these viral RNA showed no significant stimulation of their template activity for AMV reverse transcriptase. The oligo(dT)-cellulose affinity column bound neither rubella virus nor type 2 hybrid virus RNA. This suggests that both RNA genomes have a very short poly(A) tail at their 3' end. Thus, complementary DNA (cDNA) synthesis by AMV reverse transcriptase using oligo(dT) primers showed no preferential reverse transcription from the genomic 3' terminus and produced only short cDNA fragments (about 200 nucleotides). We cross-hybridized these short cDNA fragments with their viral RNA, assuming that they are copies of random sites of the genome. These cDNA-RNA hybridization analyses of physical homology between type 2 hybrid virus and rubella virus genomes revealed that about 70% of the type 2 hybrid virus genome is derived from about an 85% portion of the rubella virus genome. These values indicate that the size of the type 2 hybrid virus genome is about 21% larger than that of the rubella virus genome. Co-sedimentation studies of these viral RNA by sucrose density gradient centrifugation confirmed that the molecular weight of type 2 hybrid virus RNA is 20% higher than that of rubella virus RNA. We propose a genomic structure of the type 2 hybrid virus taking into account both physical and biochemical data.

Animals↗
PubMed1975

Virus development in enucleate cells: echovirus, poliovirus, pseudorabies virus, reovirus, respiratory syncytial virus and Semliki Forest virus.

A group of RNA viruses, echovirus, poliovirus, reovirus, respiratory syncytial virus and Semliki Forest virus have been examined for ability to grow in enucleate African green monkey kidney (BSCi) cells. Semliki Forest virus produced an almost normal yield of virus but poliovirus, echovirus, reovirus and respiratory syncytial virus, although showing clear evidence of virus replication when compared with a nuclear DNA virus (pseudorabies virus) gave much lower yields than those from nucleate cells. Analysis of enucleate cells infected with echovirus and reovirus showed no evidence of a specific block in the synthesis of any virus-specified polypeptide. Infection with vesicular stomatitis virus at intervals after enucleation demonstrated a diminishing ability to support virus growth with increasing time. It is suggested that the yield of virus obtained from an enucleate cell is related to the length of the growth cycle of the virus, the reduced yield obtained with some viruses reflecting the declining ability of the enucleate cell to support virus growth.

Autoradiography↗
PubMed1971

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. IV. Characterization of the simian virus 40 ribonucleic acid species induced by wild-type simian virus 40 and by the nondefective hybrid virus, Ad2 + ND 1 .

Ad2(+)ND(1), a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2(+)ND(1) DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2(+)ND(1) consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.

Adenoviridae↗
PubMed1992

Interactions between human immunodeficiency virus-1, hepatitis delta virus and hepatitis B virus infections in 260 chronic carriers of hepatitis B virus.

To evaluate the factors determining the severity of chronic hepatitis B virus infection and the interactions of human immunodeficiency virus and hepatitis delta virus infections, we retrospectively analyzed 260 patients, 146 of whom were followed for a mean of 31.4 +/- 1.8 mo. Human immunodeficiency virus, hepatitis B virus, and hepatitis delta virus status and aminotransferase activities, histological activity index, alcohol consumption and the prevalence of cirrhosis were investigated. The patients included 54 homosexuals, 19 parenteral drug abusers and 187 subjects with other or unidentified risk factors for exposure to hepatitis B virus. Thirty-five patients (13%) were positive for antibody to human immunodeficiency virus; 27 were homosexual and 8 were drug abusers. The mean aminotransferase activities, histological activity index and the prevalence of cirrhosis were similar in the human immunodeficiency virus-positive and human immunodeficiency virus-negative subgroups. Actuarial survival was significantly lower in the human immunodeficiency virus-negative subgroups. Actuarial survival was significantly lower in the human immunodeficiency virus-positive group than in the human immunodeficiency virus-negative subjects (p = 0.004); the cause of death was clearly related to liver failure in four of the five human immunodeficiency virus-positive patients and two of the six human immunodeficiency virus-negative subjects who died. To evaluate the factors determining the severity of liver disease, we compared homogeneous subgroups of subjects. Among the homosexual patients, the prevalence of HBeAg and hepatitis B virus DNA, aminotransferase activities and the histological activity index did not differ according to human immunodeficiency virus antibody status.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1995

A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus.

To investigate the ability of a vaccinia virus-vectored vaccine expressing the M and the S segments of Hantaan (HTN) virus (C. S. Schmaljohn, S. E. Hasty, and J. M. Dalrymple, Vaccine 10:10-13, 1992) to elicit a protective immune response against other hantaviruses, we vaccinated hamsters with the recombinant vaccine and challenged them with HTN, Seoul (SEO), or Puumala (PUU) virus. Neutralizing antibodies to HTN virus were found in all vaccinated hamsters both before and after challenge. Neutralizing antibody titers to SEO virus were present at low levels or were undetectable after two immunizations with the vaccine but were positive in all vaccinated hamsters after challenge with SEO virus and were also positive in control animals that were not challenged. Neutralizing antibodies to PUU virus were observed only in hamsters previously challenged with PUU virus. To assay for virus in the blood and tissues of the hamsters, we developed a nested reverse transcriptase (RT)-PCR with cross-reactive outer primers and serotype-specific inner primers. The RT-PCR specifically detected as little as 1 PFU of virus in serum containing high-titer neutralizing antibodies and was more sensitive than immunofluorescent antibody staining for detecting virus in lung and kidney specimens of infected hamsters. By using the RT-PCR, we found that vaccinated hamsters, challenged with HTN or SEO virus, neither were viremic nor had evidence of virus in their lungs or kidneys. In contrast, vaccinated hamsters challenged with PUU virus were viremic and had PUU virus-specific nucleic acid in their organs.

Animals↗
PubMed1986

Lymphotropic viruses, Epstein-Barr virus (EBV) and human T-cell lymphotropic virus-I (HTLV-I)/adult T-cell leukemia virus (ATLV), and HTLV-III/human immune deficiency virus (HIV) as etiological agents of malignant lymphoma and immune deficiency.

The ubiquitous, DNA herpesvirus, EBV, has B cell tropism and the geographically restricted RNA retrovirus, ATLV/HTLV-I has T cell tropism. Clinical descriptions by Burkitt and Takatsuki led to discovery of these viruses which infect silently early in life; however, ATLV is also transmitted to a spouse or by blood transfusion. In normal seropositive persons both viruses infect only 1 in about 10,000 B or T cells, respectively. EBV is associated with Burkitt's lymphoma, nasopharyngeal carcinoma, and infectious mononucleosis. ATLV is associated with adult T cell leukemia/lymphoma and smoldering T cell lymphoma. EBV infects polyclonally and is controlled by multiple cellular and humoral control mechanisms. Escape from immune surveillance as in immune deficient African children with malaria, males with x-linked lymphoproliferative syndrome, organ transplant recipients, and AIDS patients permits conversion from polyclonal to oligoclonal and finally, monoclonal malignancy. T cell immune defects permit proliferation of cells which undergo molecular and/or cytogenetic alterations. In contrast to EBV, which is integrated and nonintegrated in B cells, ATLV is monoclonally integrated. Viral transforming proteins and immune suppressive substances are produced. Immune deficiency in silent carriers of ATLV and in those with smoldering ATL suggest that immune surveillance deters emergence of ATL. Prevention of primary infection by vaccination against these lymphotropic viruses, and use of immunotherapy and antiviral drugs may potentially retard conversion of infected B or T cells to monoclonal malignancy.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Cellular proteases involved in the pathogenicity of enveloped animal viruses, human immunodeficiency virus, influenza virus A and Sendai virus.

In enveloped viruses, post-translational proteolytic activation is a critical step for the fusion activity and thus for the infectivity of the virus. In addition to the membrane receptors for the viruses, proteolytic activation is indispensable for effective virus spread in the infected host and it is a prime determinant for pathogenicity. Here we described the host cellular processing proteases, tryptase Clara and tryptase TL2, which proteolytically activate the infectivity of influenza A and Sendai viruses in the respiratory tract and HIV-1 in human CD4+ T cells, respectively. A novel trypsin-like protease, designated tryptase Clara, was purified from rat lung. The enzyme is localized in Clara cells of the bronchial epithelium and is secreted into the airway lumen. The enzyme specifically recognizes the consensus cleavage motif Gln(Glu)-X-Arg of influenza A and Sendai viruses and proteolytically activates the envelope fusion glycoproteins of the progeny viruses extracellularly in the airway lumen. Human mucus protease inhibitor and pulmonary surfactant in airway fluid inhibited the proteolytic activation of these viruses and also suppressed multiple cycles of viral replication in vitro. These results suggest that an imbalance between the amount of tryptase Clara and that of endogenous inhibitors in airway fluid is a prime determinant for pneumopathogenicity of the viruses. Therefore supplementing an endogenous inhibitor at therapeutic doses may protect against virus infection. In HIV-1 infection, binding of the gp120 envelope glycoprotein to the CD4 receptor is not sufficient in itself to allow virus entry, and an additional component(s) in the membrane is required for cell infection as a cofactor. We isolated a serine protease named tryptase TL2, in the membrane of CD4+ lymphocytes, which specifically binds to the V3 loop of HIV-1 gp120 as a cofactor. After binding, tryptase TL2 proteolytically processed gp120 into two protein species of 70 and 50 kDa and the cleavage was suppressed by a neutralizing antibody against the V3 loop. The amino acids that constitute the cleavage sites in the V3 loop of almost all HIV isolates are variable, but they are restricted to those which are susceptible to chymotryptic and/or tryptic enzyme. The multi-substrate specificity of tryptase TL2, which has tryptic and chymotryptic specificities, may correspond tot he variability of the V3 loop. The selective cleavage of the V3 loop by tryptase TL2 may lead to a conformational change of gp120, resulting in the dissociation of gp120 from gp41, exposing the fusogenic domain of the transmembrane protein gp41 following virus-host cell fusion.

Amino Acid Sequence↗
PubMed1987

A 14K envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus.

The phenomenon of genetic variability and attenuation or virulence of poxviruses is poorly understood. We have identified mutants of vaccinia virus from untreated and interferon (IFN)-treated persistently infected Friend erythroleukemia (FEL) cells that have major alterations in the size of a virus structural protein. This protein is part of the virus envelope as documented with specific monoclonal antibody (mAbC3). This protein, under reducing conditions, has a molecular weight of about 14,000 (14K) Da in wild-type virus but gained 1.5 K in mutants from untreated, persistently infected cells and about 0.5 K in mutants from IFN-treated persistently infected cells. Under nonreducing conditions, this protein forms covalently linked oligomers which also differ in size between wild-type and mutant viruses. The 14K protein elicits humoral immune response as assessed by immunoblots of two-dimensional SDS-PAGE analysis using rabbit anti-vaccinia serum. Two molecular forms of the 14K protein with different isoelectric points were found only in mutants from untreated, persistently infected cells. Protein modifications were the result of DNA sequence alterations in the virus population since the 15.5 K protein could be reverted to 14K after marker-rescue with the cloned 14K encoding gene. We provide direct evidence that changes in size of the 14K envelope protein are responsible for the small plaque size phenotype of these variants. From our previous studies (J. F. Rodriguez, R. Janeczko, and M. Esteban, 1985, J. Virol. 56, 352-356; J. F. Rodriguez, E. Paez, and M. Esteban, 1987, J. Virol. 61, 395-404) and those described here, we established that the 14K protein plays an important role in virus-host cell interactions. This is because this protein is part of the virus envelope, is highly antigenic, elicits neutralizing antibodies, has a role in virus penetration acting in cell fusion, is involved in the virus plaque size phenotype and is highly conserved among Orthopoxviruses. In addition, because mutants with altered 14K protein that have gained 1.5K in size are attenuated (S. Dallo and M. Esteban, 1987, Virology 159, 408-422), it suggests that a modified form of this protein contributes to the decrease of the virulence of vaccinia virus.

Animals↗
PubMed1993

An in-frame insertion into the Sindbis virus 6K gene leads to defective proteolytic processing of the virus glycoproteins, a trans-dominant negative inhibition of normal virus formation, and interference in virus shut off of host-cell protein synthesis.

Encoded in the genomes of all alphaviruses is a hydrophobic polypeptide of 55 amino acids, which is post-translationally modified with 4 covalently bound palmitic acids. This protein, noted as 6K, associates with membranes and is transported along with the two virus transmembranal glycoproteins to the site of virus assembly at the infected cell's plasma membrane. Previous studies showed that mutations in the 6K protein led to the slow release of aberrant, multi-cored infectious virions. In this paper, we report that an in-frame insertion of 45 nucleotides into an internal site of the 6K gene of Sindbis virus produced single-cored infectious particles at about 5% the yield of wild-type virus when the mutant was grown on avian, mammalian, and insect cells. Although the 15 amino acids were inserted at position 29 of the 55-amino-acid 6K protein, the mutation interfered with the cotranslational proteolytic processing that cleaves the 6K at its amino terminus from the Sindbis virus p62 glycoprotein and at its carboxyl terminus from the E1 glycoprotein. As a result, the amounts of normal p62 and E1 proteins were only half that made in cells infected with wild-type virus. In addition, the post-translational proteolytic conversion of p62 to E2 occurred at 10% the rate of wild-type proteins and the extensive fatty acylation normally detected on wild-type 6K protein was not found on the altered 6K protein. None of the mutated 6K protein was detected in virions, which were morphologically indistinguishable from wild-type virus. The mutant 6K virions also were similar to wild type in their rate of attachment, uncoating, and formation of an early nonstructural virus protein in avian cells. When compared with the wild-type virus, 6K29-infected cells exhibited a decreased rate of host-cell protein synthesis shut off. However, the rates of virus capsid synthesis were the same, indicating that capsid protein, per se, is not involved in shut off of host-cell protein synthesis. In complementation studies, this mutant exhibited a trans-dominant phenotype. These data provide clues about the topology of 6K protein in the membrane and its function in virus maturation.

Amino Acid Sequence↗