Complete sequence of the genome of the human isolate of Andes virus CHI-7913: comparative sequence and protein structure analysis

We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M, and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7%. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98% overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92%. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed.     

Mutations in the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein impair the incorporation of Env proteins into mature virions.

In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp41). Truncation of 147 amino acids from the carboxyl terminus of gp41 (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp41 generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp41 required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp41 did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp41 also plays a role in other steps of virus replication.     

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant.

We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.     

A study of the interaction of human spermatozoa membrane with ATP and cyclic-AMP

The three coat proteins of foot-and-mouth disease virus type O can be separated in milligram amounts using a SE-cellulose ionic exchanger, both from each other and viral RNA not quantitatively precipitated by acetic acid. The amino acid composition of the separated proteins has been determined and a statistical method has been used to calculate, for each, the most probable number of amino acids and molecular weight. The N-terminal amino acids of the three proteins are glycine, asparagine and threonine.     

The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.     

Sub-etioplast localization of the enzyme NADPH: protochlorophyllide oxidoreductase

The complete nucleotide sequence of the influenza A/PR/8/34 nucleoprotein gene was determined after cloning for dsDNA copy in pBR322. The nucleoprotein gene is 1517 nucleotides long of which 1446 nucleotides code for 482 amino acids. The calculated amino acid composition is in good agreement with those published for influenza A nucleoprotein genes. The amino acid sequence of the nucleoprotein contains clusters of basic amino acids and proline, a property shared with other nucleic-acid-associated proteins like Semliki forest virus nucleocapsid protein, VP1 protein of polyoma virus and Simian virus 40, and the core antigen of hepatitis B virus. The described nucleoprotein structure brings the number of sequenced genes of influenza A/PR/8/34 to five out of eight genes.     

Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus

Enveloped virus entry into host cells is typically initiated by an interaction between a viral envelope glycoprotein and a host cell receptor. For budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, the envelope glycoprotein GP64 is involved in host cell receptor binding, and GP64 is sufficient to mediate low-pH-triggered membrane fusion. To better define the role of GP64 in receptor binding, we generated and characterized a panel of antisera against subdomains of GP64. Eight subdomain-specific antisera were generated, and their reactivities with GP64 proteins and neutralization of virus infectivity and binding were examined. Antibodies directed against the N-terminal region of GP64 (amino acids 21 to 159) showed strong neutralization of infectivity and effectively inhibited binding of (35)S-labeled budded virions to Sf9 cells. In addition, we generated virions displaying truncated GP64 constructs. A construct displaying the N-terminal 274 amino acids (residues 21 to 294) of the ectodomain was sufficient to mediate virion binding. Additional studies of antisera directed against small subdomains revealed that an antiserum against a 40-amino-acid region (residues 121 to 160) neutralized virus infectivity. Site-directed mutagenesis was subsequently used for functional analysis of that region. Recombinant viruses expressing GP64 proteins with single amino acid substitutions within amino acids 120 to 124 and 142 to 148 replicated to high titers, suggesting that those amino acids were not critical for receptor binding or other important GP64 functions. In contrast, GP64 proteins with single amino acid substitutions of residues 153 and 156 were unable to substitute for wild-type GP64 and did not rescue a gp64 knockout virus. Further analysis showed that these substitutions substantially reduced binding of recombinant virus to Sf9 cells. Thus, the amino acid region from positions 21 to 159 was identified as a putative receptor binding domain, and amino acids 153 and 156 appear to be important for receptor binding.     

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.     

Analysis of nucleotide sequence of wheat yellow mosaic virus genomic RNAs

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNA1 is 7629 nueleotides encoding a polyprotein with 2407 amino acids, from which seven putative proteins may be produced by an autolytie cleavage processing besides the viral coat protein. The RNA2 is 3639 nueleotides and codes for a polypretein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70% and 75 % respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species within Bymovirus.     

Nucleus-targeting domain of the matrix protein (M1) of influenza virus.

The matrix protein M1 of influenza virus A/WSN/33 was shown by immunofluorescent staining to be transported into the nuclei of transfected cells without requiring other viral proteins. We postulated the existence of a potential signal sequence at amino acids 101 to 105 (RKLKR) that is required for nuclear localization of the M1 protein. When CV1 cells were transfected with recombinant vectors expressing the entire M1 protein (amino acids 1 to 252) or just the first 112 N-terminal amino acids, both the complete M1 protein and the truncated M1 protein were transported to the nucleus. In contrast, expression in CV1 cells of vectors coding for M1 proteins with deletions from amino acids 77 to 202 or amino acids 1 to 134 resulted only in cytoplasmic immunofluorescent staining of these truncated M1 proteins without protein being transported to the nucleus. Moreover, no nuclear membrane translocation occurred when CV1 cells were transfected with recombinant vectors expressing M1 proteins with deletions of amino acids 101 to 105 or with substitution at amino acids 101 to 105 of SNLNS for RKLKR. Furthermore, a synthetic oligopeptide corresponding to M1 protein amino acids 90 to 108 was also transported into isolated nuclei derived from CV1 cells, whereas oligopeptides corresponding to amino acid sequences 25 to 40, 67 to 81, and 135 to 164 were not transported into the isolated cell nuclei. These data suggest that the amino acid sequence 101RKLKR105 is the nuclear localization signal of the M1 protein.     

Norwalk virus N-terminal nonstructural protein is associated with disassembly of the Golgi complex in transfected cells

Norwalk virus is the prototype strain for members of the genus Norovirus in the family Caliciviridae, which are associated with epidemic gastroenteritis in humans. The nonstructural protein encoded in the N-terminal region of the first open reading frame (ORF1) of the Norwalk virus genome is analogous in gene order to proteins 2A and 2B of the picornaviruses; the latter is known for its membrane-associated activities. Confocal microscopy imaging of cells transfected with a vector plasmid that provided expression of the entire Norwalk virus N-terminal protein (amino acids 1 to 398 of the ORF1 polyprotein) showed colocalization of this protein with cellular proteins of the Golgi apparatus. Furthermore, this colocalization was characteristically associated with a visible disassembly of the Golgi complex into discrete aggregates. Deletion of a predicted hydrophobic region (amino acids 360 to 379) in a potential 2B-like (2BL) region (amino acids 301 to 398) near the C terminus of the Norwalk virus N-terminal protein reduced Golgi colocalization and disassembly. Confocal imaging was conducted to examine the expression characteristics of fusion proteins in which the 2BL region from the N-terminal protein of Norwalk virus (a genogroup I norovirus) or MD145 (a genogroup II norovirus) was fused to the C terminus of enhanced green fluorescent protein. Expression of each fusion protein in cells showed evidence for its colocalization with the Golgi apparatus. These data indicate that the N-terminal protein of Norwalk virus interacts with the Golgi apparatus and may play a 2BL role in the induction of intracellular membrane rearrangements associated with positive-strand RNA virus replication in cells.     

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.     

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.     

Mutational analysis of essential interactions involved in the assembly of hepatitis E virus capsid

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394-606) and p239 (amino acids 368-606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala597, Val598, Ala599, Leu601, and Ala602; furthermore, the site involved in particle formation was located at amino acids 368-394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.     

Matrix protein of Akv murine leukemia virus: genetic mapping of regions essential for particle formation.

Type C retroviruses assemble at the plasma membrane of the infected cell. Attachment of myristic acid to the N terminus of the Gag precursor polyprotein has been shown to be essential for membrane localization and virus morphogenesis. Here, we report that the matrix (MA) protein contains regions that in conjunction with myristylation are important for Gag protein stability and the assembly of murine leukemia viruses. We identified these domains by generating a series of Akv murine leukemia virus mutants carrying small in-frame deletions within the coding region of the MA protein encompassing 129 amino acids. Studies show that mutants with deletions within the segment encoding the first 102 amino acids were all replication defective, whereas the C-terminal residues 103 to 124 seem not to have any critical function in virus maturation. Cells expressing the replication-defective genomes did not release any detectable Gag proteins. In one mutant, deletion of 3 amino acids in the N terminus resulted in an inefficiently myristylated, stable Gag polyprotein. The remaining defect genomes encoded unstable Gag proteins, although they were modified with myristic acid. The results suggest that the matrix domain plays an important role in stabilizing the Gag polyprotein.     

Multiple Functions for the Basic Amino Acids of the Human T-Cell Leukemia Virus Type 1 Matrix Protein in Viral Transmission

We studied the involvement of the human T-cell leukemia virus type 1 (HTLV-1) Gag matrix protein in the cell-to-cell transmission of the virus using missense mutations of the basic amino acids. These basic amino acids are clustered at the N terminus of the protein in other retroviruses and are responsible for targeting the Gag proteins to the plasma membrane. In the HTLV–bovine leukemia virus genus of retroviruses, the basic amino acids are distributed throughout the matrix protein sequence. The HTLV-1 matrix protein contains 11 such residues. A wild-type phenotype was obtained only for mutant viruses with mutations at one of two positions in the matrix protein. The phenotypes of the other nine mutant viruses showed that the basic amino acids are involved at various steps of the replication cycle, including some after membrane targeting. Most of these nine mutations allowed normal synthesis, transport, and cleavage of the Gag precursor, but particle release was greatly affected for seven of them. In addition, four mutated proteins with correct particle release and envelope glycoprotein incorporation did not however permit cell-to-cell transmission of HTLV-1. Thus, particle release, although required, is not sufficient for the cell-to-cell transmission of HTLV-1, and the basic residues of the matrix protein are involved in steps that occur after viral particle release.     

The atmosphere of the primitive Earth and the prebiotic synthesis of amino acids

The atmosphere of the Earth at the time of its formation is now generally believed to have been reducing, an idea proposed by Oparin and extensively discussed by Urey. This atmosphere would have contained CH₄, N₂ with traces of NH₃, water and hydrogen. Only traces of NH₃ would have been present because of its solubility in water. UV light and electric discharges were the major sources of energy for amino acid synthesis, with electric discharges being the most efficient, although most other sources of energy also give amino acids.The first prebiotic electric discharge synthesis of amino acids showed that surprisingly high yields of amino acids were synthesized. Eleven amino acids were identified, four of which occur in proteins. Hydroxy acids, simple aliphatic acids and urea were also identified. These experiments have been repeated recently, and 33 amino acids were identified, ten of which occur in proteins, including all of the hydrophobic amino acids.Methionine can be synthesized by electric discharges if H₂S or CH₃SH is added to the reduced gases. The prebiotic synthesis of phenylalanine, tyrosine and trytophan involves pyrolysis reactions combined with plausible solution reactions.Eighteen amino acids have been identified in the Murchison meteorite, a type II carbonaceous chondrite, of which six occur in proteins. All of the amino acids found in the Murchison meteorite have been found among the electric discharge products. Furthermore, the ratios of amino acids in the meteorite show a close correspondence to the ratios from the electric discharge synthesis, indicating that the amino acids on the parent body of the carbonaceous chondrites were synthesized by electric discharges or by an analogous process.     

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein.

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization.     

Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes neurovirulence

Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD(50)) for neurovirulence in mice following intracerebral inoculation about 10(3)-fold lower than the LD(50) of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo.