Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2

A protein designated p14 was purified from a simian immunodeficiency virus (SIVMne) and was shown by amino acid sequence analysis to be nearly identical to the predicted translational product of a unique open reading frame (X-ORF) in the nucleotide sequences of SIVmac and human immunodeficiency virus type 2 (HIV-2). Thus the X-ORF is proven to be a new retroviral gene. The p14 is present in SIVMne in molar amounts equivalent to those of the gag proteins. This is the first example of a retrovirus that contains a substantial quantity of a viral protein that is not a product of the gag, pro, pol, or env genes. SIV p14 and its homolog in HIV-2 may function as nucleic acid binding proteins since purified p14 binds to single-stranded nucleic acids in vitro. Antisera to the purified protein detected p14 in SIVMne, SIVmac, and a homologous protein (16 kilodaltons) in HIV-2 but did not react with HIV-1. Diagnostic procedures based on this novel protein will distinguish between HIV-1 and HIV-2.     

Type-restricted neutralization of molecular clones of human immunodeficiency virus

In a study of the immunologic significance of the genetic diversity present within single isolates of human immunodeficiency virus type 1 (HIV-1), the neutralization of viruses derived from molecular clones of the HIV-1 strain HTLV-IIIB by an extensive panel of sera was compared. Sera from HIV-1-infected patients and from goats immunized with polyacrylamide gel-purified HIV-1 envelope glycoprotein (gp120), native gp120, or gp120-derived recombinant peptides, showed marked heterogeneity in neutralizing activity against these closely related viruses. The change of a single amino acid residue in gp120 may account for such "clonal restriction" of neutralizing activity.     

A single amino acid interchange yields reciprocal CTL specificities for HIV-1 gp160

For the IIIB isolate of human immunodeficiency virus type-1 (HIV-1), the immunodominant determinant of the envelope protein gp160 for cytotoxic T lymphocytes (CTLs) of H-2d mice is in a region of high sequence variability among HIV-1 isolates. The general requirements for CTL recognition of peptide antigens and the relation of recognition requirements to the natural variation in sequence of the HIV were investigated. For this purpose, a CTL line specific for the homologous segment of the envelope from the MN isolate of HIV-1 and restricted by the same class I major histocompatibility (MHC) molecule (Dd) as the IIIB-specific CTLs was raised from mice immunized with MN-env-recombinant vaccinia virus. The IIIB-specific and MN-specific CTLs were completely non-cross-reactive. Reciprocal exchange of a single amino acid between the two peptide sequences, which differed in 6 of 15 residues, led to a complete reversal of the specificity of the peptides in sensitizing targets, such that the IIIB-specific CTLs lysed targets exposed to the singly substituted MN peptide and vice versa. These data indicate the importance of single residues in defining peptide epitopic specificity and have implications for both the effect of immune pressure on selection of viral mutants and the design of effective vaccines.     

Diversity considerations in HIV-1 vaccine selection

Globally, human immunodeficiency virus-type 1 (HIV-1) is extraordinarily variable, and this diversity poses a major obstacle to AIDS vaccine development. Currently, candidate vaccines are derived from isolates, with the hope that they will be sufficiently cross-reactive to protect against circulating viruses. This may be overly optimistic, however, given that HIV-1 envelope proteins can differ in more than 30% of their amino acids. To contend with the diversity, country-specific vaccines are being considered, but evolutionary relationships may be more useful than regional considerations. Consensus or ancestor sequences could be used in vaccine design to minimize the genetic differences between vaccine strains and contemporary isolates, effectively reducing the extent of diversity by half.     

Structure and diversity of the human T-cell receptor beta-chain variable region genes

In order to characterize the variability of the expressed human T-cell receptor (TCR) beta-chain repertoire and contrast this variability to the known murine beta-chain repertoire, 15 independent complementary DNA (cDNA) clones containing TCR beta-chain variable region (V beta) genes were isolated from a human tonsil cDNA library. The nucleotide and derived amino acid sequences of these 15 V beta genes were analyzed together with 7 previously defined sequences. Fifteen different human V beta genes could be identified from 22 independent sequences. By means of DNA hybridization and sequence homology comparisons, it was possible to group these 15 genes into ten distinct V beta subfamilies, each containing from one to seven members. Minimal polymorphism was noted between individuals, except in multimember subfamilies. The amino acid sequences of these genes contain conserved amino acids that are also shared by murine TCR V beta genes and immunoglobulins; no features were found that distinguish human V beta genes from their murine counterparts. Evaluation of secondary structure showed that maximum variability coincides with generally hydrophilic portions of the amino acid sequence, while specific hydrophobic regions were conserved in all V beta genes examined.     

两株H9亚型禽流感病毒HA基因序列分析

采用Pharmacia的TimesavecDNAsynthesisKit结合RT-PCR方法，测定从鸡中的分离的2株H9亚型禽流感病毒(AIV)的HA全基因cDNA序列。结果表明这2株AIV和从香港地区鸡、鹌鹑中分离的3株H9N2亚型的AIV的亲源关系很近，HA的氨基酸同源性全部高于95％，可能是来源于同一毒株；而与2株从火鸡中分离的H9N2亚型的AIV的来源不同。     

Evidence for positive epistasis in HIV-1

Reproductive strategies such as sexual reproduction and recombination that involve the shuffling of parental genomes for the production of offspring are ubiquitous in nature. However, their evolutionary benefit remains unclear. Many theories have identified potential benefits, but progress is hampered by the scarcity of relevant data. One class of theories is based on the assumption that mutations affecting fitness exhibit negative epistasis. Retroviruses recombine frequently and thus provide a unique opportunity to test these theories. Using amino acid sequence data and fitness values from 9466 human immunodeficiency virus 1 (HIV-1) isolates, we find in contrast to these theories strong statistical evidence for a predominance of positive epistasis in HIV-1.     

Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry

Monocytes and macrophages, which may play a central role in the pathogenesis of infection with human immunodeficiency virus type 1 (HIV-1), express the CD4 molecule and Fc receptors (FcR) for immunoglobulin G (IgG). To explore the possibility that FcR mediate HIV-1 infection of monocytes, studies were conducted with the human monocytic cell line U937. These cells were exposed to HIV-1 complexed with various concentrations of serum from HIV-1 antibody-positive individuals and monitored for HIV-1 replication. Serum samples from antibody-negative normal individuals did not affect virus yields. High concentrations of antibody-positive sera showed virus-neutralizing activity; however, cells infected with HIV-1 in the presence of antibody-positive sera at subneutralizing concentrations significantly enhanced virus replication. This infection enhancement was blocked by heat-aggregated gamma-globulin. Moreover, the IgG fraction from an HIV-1 antibody-positive serum enhanced HIV-1 infection at the same serum dilution equivalents. In contrast, IgG-F(ab')2 did not enhance HIV-1 infection but showed neutralizing activity with HIV-1. These results are compatible with the concept of FcR-mediated infection enhancement and suggest that this immunological response to HIV-1, instead of protecting the host, potentially facilitates the infection.     

Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections

Efforts to solve the epidemiologic puzzle of AIDS in Africa are complicated by the presence of multiple human retroviruses. Simple serologic tests that unambiguously distinguish among infections by these retroviruses are essential. To that end, a partially conserved immunoreactive epitope was identified in the transmembrane glycoproteins of human immunodeficiency viruses (HIV) types 1 and 2. Synthetic peptides derived from these conserved domains were used in sensitive and specific immunoassays that detect antibodies in sera from patients infected with HIV-1 or HIV-2. By making single amino acid substitutions in the HIV-1 peptide, it was possible to demonstrate HIV-1 strain-specific antibody responses to this epitope. Such custom-designed peptides synthesized from this domain are likely to detect newly discovered HIV types, define infection with specific HIV strains, and allow detection of group-common antibodies.     

Single-chain antigen-binding proteins

Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.     

香蕉束顶病毒NS株系DNA组分6的克隆及序列分析

通过常规的基因克隆技术，对香蕉束顶病毒NS株系代表分离物的DNA组分6进行了克隆和序列分析，并将该分离物这个组分的序列与先前报道的广州天河分离物（属NSP株系）的该组分序列进行比较，结果表明：该组分全序列，ORF及其编码的氨基酸序列在2个分离物间的变异率分别为3.4%,17%t 2.6%,表明该组分在2个株系代表分离物间存在较显著差异，从而进一步支持了广东BBTV存在2个株系的结论，此外，NS株系代表分离物DNA组分6的全序列，ORF及其编码的氨基酸序列与澳大利亚分离物的变异率分别为14．4％，7．5％和7．1％。     

Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.     

Complete nucleotide sequences of two isolates of Cherry virus A from sweet cherry in China

Cherry virus A(CVA) is a member of the genus Capillovirus, in the family Betaflexiviridae. The infection rate of CVA was high in sweet cherry in China. We determined the complete nucleotide sequences of two isolates of CVA from Tai'an, Shandong Province, China using high fidelity PCR enzymes and specific primer pairs for amplifying long fragments in RT-PCR and RACE. The full-length sequences from isolates Ch TA11 and Ch TA12 are both 7 382 nucleotide(nt) long, excluding the poly(A) tail, encode two open reading frames(ORFs) and have similar genome organization to the two isolates in GenBank. The complete nucleotide sequence of Ch TA11 is 98.2 and 81.2% nt identity to t he isolates from Germany and India in Gen Bank, respectively, and the Ch TA12 isolate is 98.2 and 81.0% similar. Analysis of the nucleotide and amino acid sequences showed that the domain of unknown function(DUF1717) is more variable compared with other domains. This is the first report of the complete nucleotide sequences of CVA isolates infecting sweet cherry in China.     

Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies

The design of a human immunodeficiency virus-1 (HIV-1) immunogen that can induce broadly reactive neutralizing antibodies is a major goal of HIV-1 vaccine development. Although rare human monoclonal antibodies (mAbs) exist that broadly neutralize HIV-1, HIV-1 envelope immunogens do not induce these antibody specificities. Here we demonstrate that the two most broadly reactive HIV-1 envelope gp41 human mAbs, 2F5 and 4E10, are polyspecific autoantibodies reactive with the phospholipid cardiolipin. Thus, current HIV-1 vaccines may not induce these types of antibodies because of autoantigen mimicry of the conserved membrane-proximal epitopes of the virus. These results may have important implications for generating effective neutralizing antibody responses by using HIV-1 vaccines.     

Chimpanzee reservoirs of pandemic and nonpandemic HIV-1

Human immunodeficiency virus type 1 (HIV-1), the cause of human acquired immunodeficiency syndrome (AIDS), is a zoonotic infection of staggering proportions and social impact. Yet uncertainty persists regarding its natural reservoir. The virus most closely related to HIV-1 is a simian immunodeficiency virus (SIV) thus far identified only in captive members of the chimpanzee subspecies Pan troglodytes troglodytes. Here we report the detection of SIVcpz antibodies and nucleic acids in fecal samples from wild-living P. t. troglodytes apes in southern Cameroon, where prevalence rates in some communities reached 29 to 35%. By sequence analysis of endemic SIVcpz strains, we could trace the origins of pandemic (group M) and nonpandemic (group N) HIV-1 to distinct, geographically isolated chimpanzee communities. These findings establish P. t. troglodytes as a natural reservoir of HIV-1.     

Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B virus

Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.     

芜菁花叶病毒萝卜与甘蓝分离物P3基因的克隆与序列分析

芜菁花叶病毒(Turnip mosaic virus,TuMV)危害范围广,变异快,对其序列进行分析,能对病毒的演变与进化有深入的了解,为抗病育种打下基础.P3蛋白是TuMV高度容易变异的蛋白之一,包含一个对Brassica属和Raphanus属不同宿主侵染能力的决定因子,决定了宿主范围.笔者分别从北京感病的萝卜和甘蓝上分离得到4个TuMV分离物BJ - R01和BJ - 1301、BJ - B02、BJB03.利用RT - PCR克隆了这4个分离物的P3基因,测定了它们的核苷酸序列,并进行了序列分析.结果表明,4个分离物的P3基因均为1 065个碱基,编码355个氨基酸.4个分离物P3基因的同源性较高,核苷酸序列同源性在92.6％ ～99.3％,氨基酸同源性在93.5％ ～99.7％.根据系统进化树分析,4个TuMV分离物均属于world -B组.经对宿主的致病性鉴定,4个TuMV分离物均为BR致病型.4个TuMV分离物对Brassica属植物均表现出强致病性,但对Raphanus属植物致病性显现出明显的差异.