scholarly journals Positive and Negative Modulation of Virus Infectivity and Envelope Glycoprotein Incorporation into Virions by Amino Acid Substitutions at the N Terminus of the Simian Immunodeficiency Virus Matrix Protein

2003 ◽  
Vol 77 (20) ◽  
pp. 10881-10888 ◽  
Author(s):  
Julieta M. Manrique ◽  
Cristina C. P. Celma ◽  
Eric Hunter ◽  
José L. Affranchino ◽  
Silvia A. González
Keyword(s):  
Amino Acid ◽  
Matrix Protein ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Amino Acid Substitutions ◽  
Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
Amino Terminal ◽  
Env Glycoprotein

ABSTRACT The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal α-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within α-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the β-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Changes in Mumps Virus Gene Sequence Associated with Variability in Neurovirulent Phenotype

2003 ◽  
Vol 77 (21) ◽  
pp. 11616-11624 ◽  
Author(s):  
Steven A. Rubin ◽  
Georgios Amexis ◽  
Mikhail Pletnikov ◽  
Jacqueline Vanderzanden ◽  
Jeremy Mauldin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Matrix Protein ◽  
The United States ◽  
Viral Meningitis ◽  
Mumps Virus ◽  
Wild Type Virus ◽  
Neural Cells ◽  
Wild Type ◽  
Virus Variant ◽  
Vaccination Programs

ABSTRACT Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.

scholarly journals Requirement for an Aromatic Amino Acid or Histidine at the N Terminus of Sindbis Virus RNA Polymerase

1998 ◽  
Vol 72 (3) ◽  
pp. 2310-2315 ◽  
Author(s):  
Yukio Shirako ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Sindbis Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Prolonged Incubation ◽  
Rapid Degradation ◽  
N Terminus ◽  
Reticulocyte Lysates

ABSTRACT The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40°C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30°C and minute plaques at 40°C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30°C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.

scholarly journals Replication of Herpes Simplex Virus 1 Depends on the γ134.5 Functions That Facilitate Virus Response to Interferon and Egress in the Different Stages of Productive Infection

2004 ◽  
Vol 78 (14) ◽  
pp. 7653-7666 ◽  
Author(s):  
Xianghong Jing ◽  
Melissa Cerveny ◽  
Kui Yang ◽  
Bin He
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Compensatory Mutation ◽  
Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Amino Terminal ◽  
Simplex Virus

ABSTRACT The ability of the γ134.5 protein to suppress the PKR response plays a crucial role in herpes simplex virus pathogenesis. In this process, the γ134.5 protein associates with protein phosphatase 1 to form a large complex that dephosphorylates eIF-2α and thereby prevents translation shutoff mediated by PKR. Accordingly, γ134.5 null mutants are virulent in PKR-knockout mice but not in wild-type mice. However, γ134.5 deletion mutants, with an extragenic compensatory mutation, inhibit PKR activity but remain avirulent, suggesting that the γ134.5 protein has additional functions. Here, we show that a substitution of the γ134.5 gene with the NS1 gene from influenza A virus renders viral resistance to interferon involving PKR. The virus replicates as efficiently as wild-type virus in SK-N-SH and CV-1 cells. However, in mouse 3T6 cells, the virus expressing the NS1 protein grows at an intermediate level between the wild-type virus and the γ134.5 deletion mutant. This decrease in growth, compared to that of the wild-type virus, is due not to an inhibition of viral protein synthesis but rather to a block in virus release or egress. Virus particles are predominantly present in the nucleus and cytoplasm. Notably, deletions in the amino terminus of the γ134.5 protein lead to a significant decrease in virus growth in mouse 3T6 cells, which is independent of eIF-2α dephosphorylation. In correlation, a series of deletions in the amino-terminal domain impair nuclear as well as cytoplasmic egress. These results indicate that efficient viral replication depends on the γ134.5 functions required to prevent the PKR response and to facilitate virus egress in the different stages during virus infection.

scholarly journals Cleavage of Human Cytomegalovirus Protease pUL80a at Internal and Cryptic Sites Is Not Essential but Enhances Infectivity

2005 ◽  
Vol 79 (20) ◽  
pp. 12961-12968 ◽  
Author(s):  
Amy N. Loveland ◽  
Chee-Kai Chan ◽  
Edward J. Brignole ◽  
Wade Gibson
Keyword(s):  
Human Cytomegalovirus ◽  
Cleavage Site ◽  
Biological Significance ◽  
Virus Production ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Biological Importance

ABSTRACT The cytomegalovirus (CMV) maturational protease, assemblin, contains an “internal” (I) cleavage site absent from its homologs in other herpesviruses. Blocking this site for cleavage did not prevent replication of the resulting I− mutant virus. However, cells infected with the I− virus showed increased amounts of a fragment produced by cleavage at the nearby “cryptic” (C) site, suggesting that its replication may bypass the I-site block by using the C site as an alternate cleavage pathway. To test this and further examine the biological importance of these cleavages, we constructed two additional virus mutants—one blocked for C-site cleavage and another blocked for both I- and C-site cleavage. Infectivity comparisons with the parental wild-type virus showed that the I− mutant was the least affected for virus production, whereas infectivity of the C− mutant was reduced by ≈40% and when both sites were blocked virus infectivity was reduced by nearly 90%, providing the first evidence that these cleavages have biological significance. We also present and discuss evidence suggesting that I-site cleavage destabilizes assemblin and its fragments, whereas C-site cleavage does not.

scholarly journals Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil α-Helix Important for Core Assembly

2001 ◽  
Vol 75 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rushika Perera ◽  
Katherine E. Owen ◽  
Timothy L. Tellinghuisen ◽  
Alexander E. Gorbalenya ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Wild Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
In Vitro Assembly ◽  
Α Helix

ABSTRACT The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an α-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with β-branched residues and conserved leucine residues occupying the a andd positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.

scholarly journals Nonstructural Protein σ1s Mediates Reovirus-Induced Cell Cycle Arrest and Apoptosis

2013 ◽  
Vol 87 (23) ◽  
pp. 12967-12979 ◽  
Author(s):  
Karl W. Boehme ◽  
Katharina Hammer ◽  
William C. Tollefson ◽  
Jennifer L. Konopka-Anstadt ◽  
Takeshi Kobayashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Nonstructural Protein ◽  
Wild Type Virus ◽  
Type Virus ◽  
Null Mutant ◽  
Wild Type ◽  
Induce Cell Cycle Arrest ◽  
Amino Terminal ◽  
Cycle Arrest

Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuatedin vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.

scholarly journals The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

2016 ◽  
Vol 90 (23) ◽  
pp. 10612-10628 ◽  
Author(s):  
Chetan D. Meshram ◽  
Pradyumna S. Baviskar ◽  
Cherie M. Ognibene ◽  
Antonius G. P. Oomens
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Surface ◽  
Fusion Protein ◽  
Matrix Protein ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Virus Like Particle ◽  
Syncytial Virus

ABSTRACTVirus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology.IMPORTANCEhRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.

The mechanism of antigenic drift in influenza virus: sequence changes in the haemagglutinin of variants selected with monoclonal hybridoma antibodies

1980 ◽  
Vol 288 (1029) ◽  
pp. 313-326 ◽  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Monoclonal Antibodies ◽  
Hong Kong ◽  
Amino Acid ◽  
The Other ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Sequence Change

Antigenic variants of the A/PR8 (H0N1) and A/Hong Kong/68 (H3N2) strains of influenza virus were isolated after a single passage of these viruses in the presence of monoclonal hybridoma antibodies to the haemagglutinin. Hyperimmune rabbit antisera reacted (in haemagglutination-inhibition tests) to high titre with both wild-type and variant viruses, but the monoclonal antibodies, which reacted with the wild-type virus to titres of the order of 1/10 5 did not react at all (or to very low titre) with the variants that they selected. This suggests that the changes occurring in the monoclonal variants are restricted to a single antigenic site out of many on the haemagglutinin molecule. Amino acid analysis of the soluble tryptic peptides from the haemagglutinin ‘spikes’ of wild-type and variant viruses suggest that the dramatic loss in the ability of the variants to bind the monoclonal antibody used in their selection is associated with a single change in the amino acid sequence of the large haemagglutinin polypeptide, HA 1 . For PR8 virus, eight out of ten variants selected with one monoclonal antibody showed the same sequence change of serine to leucine in the HA 1 polypeptide. The change in the other two variants was not determined. No sequence data on PR8 haemagglutinin are available, so the experiments were continued with a Hong Kong (H3N2) strain where much of the sequence of HA 1 and HA 2 is known. Three different monoclonal hybridoma antibodies to A/Mem/1/71 (H3N2) haemagglutinin were used to select a total of ten variants of this virus. Variants selected with one monoclonal antibody were not recognized by the other two monoclonal antibodies as being different from wild-type virus, suggesting that the three antibodies bound to different sites on the surface of the haemagglutinin molecules. Each of the variants occurred with a frequency of about 1 in 105 in the wild-type virus. One group of our variants selected with H14/A2 monoclonal antibody showed the same antigenic properties and the same sequence change (asparagine to lysine) in the N-terminal half of HA 1 . Of three variants selected with H14/A20, two showed a different change at a locus also in the N-terminal region of HA 1 (a proline was replaced by serine in one variant and by leucine in the other). Of the other three variants (selected with H14/A21 monoclonal antibody) one showed a change in HAX of serine to tyrosine. This change occurred in residue number 37 of cyanogen bromide fragment 2 (CN2). In the other two variants the change in HAX has not been determined, but in these a tryptic peptide comprising residues 49-56 of CN2 was missing. The tryptic peptides of the HA 1 polypeptide, showing changes in the variants selected with monoclonal antibodies, were also found to undergo sequence changes in naturally occurring Hong Kong variants isolated from man. In each case, however, the sequence changes in the monoclonal variants were different from those in the field strains. No changes were found in the HA 2 polypeptide from any of the variants.

scholarly journals Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission

2007 ◽  
Vol 88 (6) ◽  
pp. 1821-1830 ◽  
Author(s):  
Igor B. Kaplan ◽  
Lawrence Lee ◽  
Daniel R. Ripoll ◽  
Peter Palukaitis ◽  
Frederick Gildow ◽  
...  
Keyword(s):  
Structural Model ◽  
Point Mutations ◽  
Potato Leafroll Virus ◽  
Primary Component ◽  
Wild Type Virus ◽  
Individual Plant ◽  
Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
Leafroll Virus

The coat protein (CP) of potato leafroll virus (PLRV) is the primary component of the capsid, and is a multifunctional protein known to be involved in vector transmission and virus movement within plant hosts, in addition to particle assembly. Thirteen mutations were generated in various regions of the CP and tested for their ability to affect virus–host and virus–vector interactions. Nine of the mutations prevented the assembly of stable virions. These mutants were unable to infect systemically four different host species. Furthermore, although virus replication and translation of the CP were similar for the mutants and wild-type virus in individual plant cells, the translation of the CP readthrough product was affected in several of the mutants. Four of the mutants were able to assemble stable particles and infect host plants systemically, similarly to the wild-type virus; however, two of the mutants were transmitted less efficiently by aphid vectors. Based on a computer-generated model of the PLRV CP, the mutations that prevented virion assembly were associated with subunit interfaces, while the amino acid alterations in the assembly-competent mutants were associated with surface loops. This and previous work indicates that the CP structural model has value in predicting the structural architecture of the virion.

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