Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of nonstructural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells.

Virus-specific tubules are characteristic of orbivirus infections and are likely to play an important role in virus morphogenesis. It has been shown that for bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae, the virus-encoded NS1 protein forms tubules in insect cells when the BTV segment M6 gene is expressed by using a baculovirus vector. To understand the function of NS1 tubules and to identify the sequences involved in their polymerization, a series of mutant NS1 genes was generated and expressed in insect cell cultures by using baculovirus vectors. Three of the mutants were deletion mutants. One (AcNS1.dNT10) lacked 10 of the amino-terminal amino acids, and the other two mutants (AcNS1.dCT20 and AcNS1.dCT43) lacked 20 or 43 of the carboxy-terminal amino acids. In addition, site-directed mutants were constructed in which various single cysteines or pairs of cysteines were changed to serines. The ability of each mutant protein to form tubules was investigated. None of the deletion mutants formed tubules. The constructs in which the cysteines at amino acid positions 337 and/or 340 were replaced by serines (e.g., AcNS1.C337S,C340S) also did not form tubules. Instead, the NS1 protein of these and the deletion mutants made ribbon-like structures which formed large aggregates. Mutations involving six other cysteines (i.e., AcNS1.C37S,C43S,AcNS1.C462S,C465S, AcNS1.C104S, and AcNS1.C364S) produced tubules. The results show that both the amino and carboxy termini of the NS1 protein molecule and the cysteines at residues 337 and 340 are essential for tubule formation.     

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Deletion and mutational analyses of bluetongue virus NS2 protein indicate that the amino but not the carboxy terminus of the protein is critical for RNA-protein interactions.

Genome segment 8 (S8) of bluetongue virus serotype 10 (BTV-10) encodes the nonstructural protein NS2. This protein, which has single-stranded RNA (ssRNA) binding capacity, is found in BTV-infected cells in the form of virus inclusion bodies (VIBs). To identify the domain(s) important for RNA binding and oligomerization of the protein, a number of deletions were made in regions of the gene that code for either the amino or carboxy terminus of the protein. The modified genes were cloned into and expressed from baculovirus vectors based on Autographa californica nuclear polyhedrosis virus. Truncated NS2 proteins were individually analyzed for the ability to bind ssRNA and to form VIBs. The results indicated that the carboxy terminus of the protein is involved neither in RNA binding nor in the formation of VIBs. The amino terminus of NS2 was shown to be essential for ssRNA binding but not for NS2 protein oligomerization. Point mutations that involved the substitution of various charged residues at the amino terminus of NS2 were generated and tested for the ability to bind ssRNA. The results showed that the arginines at amino acid residues 6 and 7 and the lysine at residue 4, but not the glutamic acid at residue 2, are involved in ssRNA binding.     

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.     

Functional replacement of the carboxy-terminal two-thirds of the influenza A virus NS1 protein with short heterologous dimerization domains

The NS1 protein of influenza A/WSN/33 virus is a 230-amino-acid-long protein which functions as an interferon alpha/beta (IFN-alpha/beta) antagonist by preventing the synthesis of IFN during viral infection. In tissue culture, the IFN inhibitory function of the NS1 protein has been mapped to the RNA binding domain, the first 73 amino acids. Nevertheless, influenza viruses expressing carboxy-terminally truncated NS1 proteins are attenuated in mice. Dimerization of the NS1 protein has previously been shown to be essential for its RNA binding activity. We have explored the ability of heterologous dimerization domains to functionally substitute in vivo for the carboxy-terminal domains of the NS1 protein. Recombinant influenza viruses were generated that expressed truncated NS1 proteins of 126 amino acids, fused to 28 or 24 amino acids derived from the dimerization domains of either the Saccharomyces cerevisiae PUT3 or the Drosophila melanogaster Ncd (DmNcd) proteins. These viruses regained virulence and lethality in mice. Moreover, a recombinant influenza virus expressing only the first 73 amino acids of the NS1 protein was able to replicate in mice lacking three IFN-regulated antiviral enzymes, PKR, RNaseL, and Mx, but not in wild-type (Mx-deficient) mice, suggesting that the attenuation was mainly due to an inability to inhibit the IFN system. Remarkably, a virus with an NS1 truncated at amino acid 73 but fused to the dimerization domain of DmNcd replicated and was also highly pathogenic in wild-type mice. These results suggest that the main biological function of the carboxy-terminal region of the NS1 protein of influenza A virus is the enhancement of its IFN antagonist properties by stabilizing the NS1 dimeric structure.     

Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein.

The most abundant Epstein-Barr virus mRNA in a latently infected cell line, IB4, established by in vitro growth transformation with virus, was a 2,8-kilobase RNA encoded by largely unique DNA near the right end of the genome. The RNA was transcribed from right to left, and two introns were spliced out. This region of the genome was sequenced, and the exons of the RNA were identified by S1 analysis of DNA-RNA hybrids and primer extension. The first start codon in the RNA was 40 nucleotides from its 5' end. Beginning with the start codon, there was a 1,158-nucleotide open reading frame which crossed both introns. The important characteristics of the translated protein were as follows. (i) The amino terminus was highly charged and not suggestive of a leader sequence. (ii) There were six markedly hydrophobic alpha-helical domains, each having 21 amino acids and connected by 5 to 7 amino acid segments predicted to be reverse turns. (iii) The carboxy-terminal 200 amino acids were markedly acidic, containing 6 glutamic and 37 aspartic acids. The hydrophobic region is predicted to form six membrane-spanning regions, leaving the short charged amino terminus and long acidic carboxy terminus on the inside of the membrane. This protein could be responsible for the new antigen detected in the plasma membrane of Epstein-Barr virus-transformed cells, lymphocyte-determined membrane antigen. There were two other open reading frames in the RNA.     

The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation.

A collection of C-terminal deletion mutants of the influenza A virus NS1 gene has been used to define the regions of the NS1 protein involved in its functionality. Immunofluorescence analyses showed that the NS1 protein sequences downstream from position 81 are not required for nuclear transport. The capacity of these mutants to bind RNA was studied by in vitro binding tests using a model vRNA probe. These experiments showed that the N-terminal 81 amino acids of NS1 protein are sufficient for RNA binding activity. The collection of mutants also served to map the NS1 sequences required for nuclear retention of mRNA and for stimulation of viral mRNA translation, using the NP gene as reporter. The results obtained indicated that the N-terminal 113 amino acids of NS1 protein are sufficient for nuclear retention of mRNA and stimulation of viral mRNA translation. The possibility that this region of the protein may be sufficient for virus viability is discussed in relation to the sequences of NS1 genes of field isolates and to the phenotype of known viral mutants affected in the NS1 gene.     

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.     

Characterization of tubule and monomer derived from VP4 protein of infectious bursal disease virus

The VP4 protein of infectious bursal disease virus (IBDV) is a serine protease that processes the polyprotein for viral assembly. VP4 has been found to associate primarily with type II IBDV tubules that are 24 nm in diameter. In this study, a chimeric VP4, assigned as HS1VP4, was constructed with a VP4-autocleavage site inserted between the N-terminal His-tag and the VP4 sequence. The results showed that the VP4 forms tubules after the self-cleavage of HS1VP4 when expressed in Escherichia coli. Furthermore, a deletion of 28 amino acids at the C-terminus of VP4 resulted in monomers and dimers instead of tubule formation; mutants of S652A and K692A at active site destroyed the activity. The endopeptidase activity of these monomers and dimers was approximately 12.5 times higher than that of VP4 tubules. Additionally, the formation of tubules inhibited VP4 protease activity, as demonstrated through in vitro assays. The production and characterization of monomers or dimers that have greater endopeptidase activity and protease activity than tubules can provide further insight into VP4 tubule assembly and the regulation of VP4 activity in host cells; this insight will facilitate the development of new anti-IBDV strategies.     

Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein 2 at multiple sites.

Three polymerase proteins of influenza type A virus interact with each other to form the active polymerase complex. Polymerase basic protein 1 (PB1) can interact with PB2 in the presence or absence of polymerase acidic protein. In this study, we investigated the domains of PB1 involved in complex formation with PB2 in vivo, using coexpression and coimmunoprecipitation of the PB1-PB2 complex with monospecific antibodies. Results show that PB1 possesses at least two regions which can interact independently and form stable complexes with PB2. Both of these regions are located at the NH2 terminus of PB1; the COOH-terminal half of PB1 is not involved in interacting with PB2. Deletion analysis further demonstrated that the interacting regions of PB1 encompass amino acids (aa) 48 to 145 and aa 251 to 321. Linker insertions throughout the PB1 sequences did not affect complex formation with PB2. Deletion and linker-insertion mutants of PB1 were tested for polymerase activity in vivo. For this analysis, we developed a simplified assay for viral polymerase activity that uses a reporter chloramphenicol acetyltransferase gene containing the 5' and 3' ends of influenza viral promoter and nontranslating regions (minus sense) of the NS gene joined to a hepatitis delta virus ribozyme at its 3' end. This assay demonstrated that all deletion mutants of PB1 exhibited either background or greatly reduced polymerase activity irrespective of the ability to interact with PB2 and that all linker-insertion mutants except one at the extreme COOH end (L-746) of PB1 were also negative for viral polymerase activity. These results show that compared with complex formation of PB1 with PB2, the polymerase activity of PB1 was extremely sensitive to structural perturbation.     

Two nuclear location signals in the influenza virus NS1 nonstructural protein.

The NS1 protein of influenza A virus has been shown to enter and accumulate in the nuclei of virus-infected cells independently of any other influenza viral protein. Therefore, the NS1 protein contains within its polypeptide sequence the information that codes for its nuclear localization. To define the nuclear signal of the NS1 protein, a series of recombinant simian virus 40 vectors that express deletion mutants or fusion proteins was constructed. Analysis of the proteins expressed resulted in identification of two regions of the NS1 protein which affect its cellular location. Nuclear localization signal 1 (NLS1) contains the stretch of basic amino acids Asp-Arg-Leu-Arg-Arg (codons 34 to 38). This sequence is conserved in all NS1 proteins of influenza A viruses, as well as in that of influenza B viruses. NLS2 is defined within the region between amino acids 203 and 237. This domain is present in the NS1 proteins of most influenza A virus strains. NLS1 and NLS2 contain basic amino acids and are similar to previously defined nuclear signal sequences of other proteins.     

Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface

Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.     

Mutational analysis of the herpes simplex virus triplex protein VP19C

Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal approximately 100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.     

A single point mutation in the VP7 major core protein of bluetongue virus prevents the formation of core-like particles.

To understand the assembly process of bluetongue virus (BTV), we have established a functional assay which allows us to produce and manipulate BTV core-like particles (CLPs) composed of the viral VP7 and VP3 proteins. A cDNA clone encoding the 349-amino-acid VP7 protein has been manipulated to generate deletion, extension, and site-specific mutants. Each mutant was coexpressed with the BTV VP3 protein to generate CLPs. Deletion and extension mutants involving the VP7 carboxy terminus prevented CLP formation, while an extension mutant involving an 11-amino-acid rabies virus sequence added to the amino terminus of VP7 allowed CLP formation. Substitution of either of two cysteine residues of VP7 (Cys-15 or Cys-65) by serine also did not prevent CLP formation; however, substitution of the single lysine residue of VP7 (Lys-255) by leucine abrogated CLP formation, indicating a critical role for this lysine.     

Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus.

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.     

Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells]

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

Nuclear localization of the PEP protein tyrosine phosphatase.

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.