Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission

BackgroundThe human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) plays a pivotal role in the viral lifecycle: including encapsulating the viral genome, aiding in strand transfer during reverse transcription, and packaging two copies of the viral genome into progeny virions. Another gag gene product, p6, plays an integral role in successful viral budding from the plasma membrane and inclusion of the accessory protein Vpr within newly budding virions. In this study, we have characterized the gag NC and p6 genes from six mother-infant pairs following vertical transmission by performing phylogenetic analysis and by analyzing the degree of genetic diversity, evolutionary dynamics, and conservation of functional domains.ResultsPhylogenetic analysis of 168 gag NC and p6 genes sequences revealed six separate subtrees that corresponded to each mother-infant pair, suggesting that epidemiologically linked individuals were closer to each other than epidemiologically unlinked individuals. A high frequency (92.8%) of intact open reading frames of NC and p6 with patient and pair specific sequence motifs were conserved in mother-infant pairs' sequences. Nucleotide and amino acid distances showed a lower degree of viral heterogeneity, and a low degree of estimates of genetic diversity was also found in NC and p6 sequences. The NC and p6 sequences from both mothers and infants were found to be under positive selection pressure. The two important functional motifs within NC, the zinc-finger motifs, were highly conserved in most of the sequences, as were the gag p6 Vpr binding, AIP1 and late binding domains. Several CTL recognition epitopes identified within the NC and p6 genes were found to be mostly conserved in 6 mother-infant pairs' sequences.ConclusionThese data suggest that the gag NC and p6 open reading frames and functional domains were conserved in mother-infant pairs' sequences following vertical transmission, which confirms the critical role of these gene products in the viral lifecycle.

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title: THE VERTICAL TRANSMISSION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1: Molecular and Biological Properties of the Virus
abstract: The vertical (mother-to-infant) transmission of human immunodeficiency virus type 1 (HIV-1) occurs at an estimated rate of more than 30% and is the major cause of AIDS in children. Numerous maternal parameters, including advanced clinical stages, low CD4+ lymphocyte counts, high viral load, immune response, and disease progression have been implicated in an increased risk of vertical transmission. While the use of antiretroviral therapy (ART) during pregnancy has been shown to reduce the risk of vertical transmission, selective transmission of ART-resistant mutants has also been documented. Elucidation of the molecular mechanisms of vertical transmission might provide relevant information for the development of effective strategies for prevention and treatment. By using HIV-1 infected mother-infant pairs as a transmitter-recipient model, the minor genotypes of HIV-1 with macrophage-tropic and non-syncytium-inducing phenotypes (R5 viruses) in infected mothers were found to be transmitted to their infants and were initially maintained in the infants with the same properties. In addition, the transmission of major and multiple genotypes has been suggested. Furthermore, HIV-1 sequences found in non-transmitting mothers (mothers who failed to transmit HIV-1 to their infants in the absence of ART) were less heterogeneous than those from transmitting mothers, suggesting that viral heterogeneity may play an important role in vertical transmission. In the analysis of other regions of the HIV-1 genome, we have shown a high conservation of intact and functional gag p17, vif, vpr, vpu, tat, and nef open reading frames following mother-to-infant transmission. Moreover, the accessory genes, vif and vpr, were less functionally conserved in the isolates of non-transmitting mothers than transmitting mothers and their infants. We, therefore, should target the properties of transmitted viruses to develop new and more effective strategies for the prevention and treatment of HIV-1 infection.

title: Maternal-Fetal Transmission of Human Immunodeficiency Virus.
abstract: The World Health Organization estimates that by year 2000, 10 million children will be infected with human immunodeficiency virus type 1 (HIV-1) at birth and will subsequently develop AIDS. Perinatally acquired infections account for the majority of all HIV-1 cases in children, with an estimated mother-to-infant transmission rate of more than 30%. It is not clear why more than half of the children born to HIV-1-infected mothers are uninfected. Maternal transmission of HIV-1 occurs at three levels: prepartum, intrapartum, and postpartum. Several maternal parameters including advanced clinical stages of the mother, low CD4+ lymphocyte counts, maternal immune response to HIV-1, recent infection, high level of circulating HIV-1, and maternal disease progression have been implicated in an increased risk of mother-to-infant transmission of HIV-1. Viral factors influencing mother-to-infant transmission are not known. Furthermore, several other factors such as acute infection during pregnancy, presence of other sexually transmitted diseases (STD) or other chronic infections, vaginal bleeding, disruption of placental integrity, premature rupture of membrane (PROM), and preterm PROM have been associated with mother-to-infant transmission of HIV-1. In addition, tobacco and cigarette smoking during pregnancy have been shown to triple the rate of maternal transmission of HIV-1. The AIDS Clinical Trial Group (ACTG) suggested that zidovudine (ZDV) can reduce therate of mother-to-infant transmission of HIV-1 if administered to HIV-1-infected pregnant women with CD4 counts greater than 200. Moreover, this study failed to take into consideration several factors that may influence maternal transmission of HIV-1. However, the molecular mechanisms involved in mother-to-infant transmission of HIV-1 are not understood, which makes it more difficult to define strategies for effective treatment and prevention of HIV-1 infection in children. Several groups are engaged in the understanding of the molecular and biological properties of HIV-1 influencing mother-to-infant transmission. Results from my and several other laboratories suggest that the minor genotypes, subtypes, or variants of HIV-1 found in a genetically heterogeneous virus population of infected mothers are transmitted to their infants. The minor HIV-1 genotype predominates initially as a homogeneous population in the infant and then becomes diverse as the infant matures. Furthermore, transmission of a major or multiple HIV-1 genotypes from mother to infant has been reported. Taken together, these results strongly suggest that there are differences among the molecular and biological properties of the maternal variants that are transmitted to the infants and the maternal variants that are not transmitted to the infants. The understanding of the molecular and biological properties of the transmitted viruses will enable researchers to target a particular subtype in the mothers that is transmitted to the infants.

title: A prospective study of infants born to women seropositive for human immunodeficiency virus type 1
abstract: Assessment of the risks of transmission of infection with human immunodeficiency virus type 1 (HIV-1) from mother to newborn is difficult, partly because of the persistence for up to a year of maternal antibodies transmitted passively to the infant. To determine the frequency of perinatal transmission of HIV infection, we studied from birth 308 infants born to seropositive women, 62 percent of whom were intravenous drug abusers. Of 117 infants evaluated 18 months after birth, 32 (27 percent) were seropositive for HIV or had died of the acquired immunodeficiency syndrome (AIDS) (n = 6); of the 32, only 2 remained asymptomatic. Another 76 infants (65 percent) were seronegative and free of symptoms, whereas 9 (8 percent) were seronegative but had symptoms suggestive of HIV-1 infection. The infants infected with HIV-1 did not differ from the others at birth with respect to weight, height, head circumference, or rate of malformations, but as compared with newborns who were seronegative at 18 months, their serum IgM levels were higher (78 +/- 81 mg per deciliter vs. 38 +/- 39 mg per deciliter; P less than 0.03) and their CD4 lymphocyte counts were lower (2054 +/- 1221 per cubic millimeter vs. 2901 +/- 1195 per cubic millimeter; P less than 0.006). Neither maternal risk factors nor the route of delivery was a predictor of seropositivity at 18 months; however, 5 of the 6 infants who were breast-fed became seropositive, as compared with 25 of 99 who were not (P less than 0.01). We conclude that approximately one third of the infants born to seropositive mothers will have evidence of HIV-1 infection or of AIDS by the age of 18 months, and that about one fifth of this group will have died.

title: Infants born to mothers seropositive for human immunodeficiency virus. Preliminary findings from a multicentre European study
abstract: present study was to evaluate whether the induction of multiple ovulation can increase the pregnancy rates in couples treated with AIH. Multiple follicular development was induced by means of clomiphene citrate (CC) plus purified folliclestimulating hormone (FSH) in 17 couples undergoing AIH for male-related or unexplained infertility. In spite of the long duration of infertility (> 6 years), 10 couples (58.8%) achieved pregnancy within 6 months of treatment. This figure was significantly higher than that obtained in 120 couples who underwent AIH either during spontaneous cycles or after induction of follicular development with CC alone. These results suggest that the availability of multiple mature oocytes increases the conception rate during each cycle, as reported for in vitro fertilization (IVF) programs. It is therefore suggested that a trial of induction of multiple follicular development be performed in couples with male-related or unexplained infertility before their inclusion in protocols for invasive procedures, such as IVF or gamete intrafallopian transfer.

title: Perinatal transmission of the human immunodeficiency virus type 1 to infants of seropositive women in Zaire.
abstract: To examine perinatal transmission of the human immunodeficiency virus type 1 (HIV-1) in Zaire, we screened 8108 women who gave birth at one of two Kinshasa hospitals that serve populations of markedly different socioeconomic status. For up to one year, we followed the 475 infants of the 466 seropositive women (5.8 percent of those screened) and the 616 infants of 606 seronegative women matched for age, parity, and hospital. On the basis of clinical criteria, 85 of the seropositive women (18 percent) had the acquired immunodeficiency syndrome (AIDS). The infants of seropositive mothers, as compared with those of seronegative mothers, were more frequently premature, had lower birth weights, and had a higher death rate in the first 28 days (6.2 vs. 1.2 percent; P less than 0.0001). The patterns were similar at the two hospitals. Twenty-one percent of the cultures for HIV-1 of 92 randomly selected cord-blood samples from infants of seropositive women were positive. T4-cell counts were performed in 37 seropositive women, and cord blood from their infants was cultured. The cultures were positive in the infants of 6 of the 18 women with antepartum T4 counts of 400 or fewer cells per cubic millimeter, as compared with none of the infants of the 19 women with more than 400 T4 cells per cubic millimeter (P = 0.02). One year later, 21 percent of the infants of the seropositive mothers had died as compared with 3.8 percent of the control infants (P less than 0.001), and 7.9 percent of their surviving infants had AIDS. We conclude that the mortality rates among children of seropositive mothers are high regardless of socioeconomic status, and that perinatal transmission of HIV-1 has a major adverse effect on infant survival in Kinshasa.

title: POSTNATAL TRANSMISSION OF HIV FROM MOTHER TO CHILD
abstract: 2 cases of post-partum transmission of human immunodefficiency virus (HIV) from mother to infant from Kigali Rwanda are reported. All results were confirmed by western blot tests. 1) A 23-year-old woman with no history of extramarital sexual contact blood transfusions drug abuse or injections during pregnancy experienced severe bleeding during childbirth and was transfused with 2 units blood one of which had been drawn from a donor who later proved to be seropositive. The child sickened and died at 19 months of chronic diarrhea after having presented with failure to thrive generalized lymphadenopathy oral thrush and hepatomegaly moderate psychomotor delay and lobar pneumonia on chest x-ray. At 15 months post-partum the mother showed mild lymphocytosis and had antibodies to HIV. She had no dermatitis and breast-fed the child without problems until she died. 2) The 2nd case of a boy was quite similar: a postpartum transfusion from a donor who later prove to be seropositive was the most likely source of the mothers HIV infection. The child in question was still being breast fed at 15 months having begun to show symptoms of HIV infection at 5 months of age. A 30-month-old sibling was thriving and seronegative. In both cases the fathers were seronegative. Given the length of time the children had been breast fed and past studies showing the presence of HIV in breast milk as well as colostrum breast milk appears to be the most likely source of the infection in these cases. They were 2 cases out of 107 pediatric cases of AIDS and AIDS-related complex studied over 2 years. The results do not indicate a need to discourage mothers from breastfeeding but do underline the need to enforce measures to avoid contamination of banked breast milk.

title: Mother-to-child transmission of HIV infection. The European Collaborative Study.
abstract: 271 children born to HIV-infected mothers in 8 European centres are being followed up from birth in a multicentre, collaborative study. By June, 1988, 45% had been followed for over 1 year: 10 had developed AIDS or AIDS-related complex, all by the age of 9 months, of whom 5 had died. 22 other children had symptoms or signs suggestive of HIV infection; of these, 12 had immunological abnormalities, 9 of whom were infected. 5 children had problems not related to HIV, including 3 neonatal deaths. The other 234 children are immunologically normal and clinically well. The median age of antibody loss was 10.3 months, although 1 did not lose antibody until over 18 months. None lost antibody and then became and remained seropositive. Of 100 children followed for more than 15 months, 19 had persistent antibody, and 5 were antibody-negative but presumed to be infected because of virus isolation or antigen detection; these 5 children were clinically and immunologically normal. The estimated vertical transmission rate was 24%.

title: Genetic analysis of human immunodeficiency virus type 1 envelope V3 region isolates from mothers and infants after perinatal transmission
abstract: The human immunodeficiency virus type 1 (HIV-1) sequences from variable region 3 (V3) of the envelope gene were analyzed from seven infected mother-infant pairs following perinatal transmission. The V3 region sequences directly derived from the DNA of the uncultured peripheral blood mononuclear cells from infected mothers displayed a heterogeneous population. In contrast, the infants' sequences were less diverse than those of their mothers. In addition, the sequences from the younger infants' peripheral blood mononuclear cell DNA were more homogeneous than the older infants' sequences. All infants' sequences were different but displayed patterns similar to those seen in their mothers. In the mother-infant pair sequences analyzed, a minor genotype or subtype found in the mothers predominated in their infants. The conserved N-linked glycosylation site proximal to the first cysteine of the V3 loop was absent only in one infant's sequence set and in some variants of two other infants' sequences. Furthermore, the HIV-1 sequences of the epidemiologically linked mother-infant pairs were closer than the sequences of epidemiologically unlinked individuals, suggesting that the sequence comparison of mother-infant pairs done in order to identify genetic variants transmitted from mother to infant could be performed even in older infants. There was no evidence for transmission of a major genotype or multiple genotypes from mother to infant. In conclusion, a minor genotype of maternal virus is transmitted to the infants, and this finding could be useful in developing strategies to prevent maternal transmission of HIV-1 by means of perinatal interventions.

title: Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants.
abstract: Multiple human immunodeficiency virus type-1 sequences from the V3 and V4-V5 regions of the envelope gene were analyzed from three mother-infant pairs. The infants' viral sequences were less diverse than those of their mothers. In two pairs, a proviral form infrequently found in the mother predominated in her infant. A conserved N-linked glycosylation site within the V3 region, present in each mother's sequence set, was absent in all of the infants' sequence sets. These findings demonstrate that a minor subset of maternal virus is transmitted to the infant.

title: Biological characterization of HIV type 1 envelope V3 regions from mothers and infants associated with perinatal transmission.
abstract: Our previous study has shown that the human immunodeficiency virus type 1 (HIV-1) envelope V3 region minor genotypes of infected mothers were transmitted to their infants and predominated initially as a homogeneous virus population in the infants (Ahmad N, Baroudy BM, Baker RC, et al.: J Virol 1995;69:1001-1012). Here we have characterized the biological properties, including cellular tropism, replication efficiency, cytopathic effects, and coreceptor utilization, of these V3 region isolates from mothers and infants. Nineteen V3 region sequences from three mother-infant pairs, including the minor variants of mothers and the major variants of infants as characterized in our previous study, were reciprocally inserted into an HIV-1 infectious molecular clone, pNL4-3, and chimeric viruses were generated by DNA transfections into HeLa cells. Equal amounts of chimeric viruses were then used to infect T lymphocyte cell lines (A3.01 and MT-2), primary blood lymphocytes (PBLs), primary monocyte-derived macrophages (MDMs), and coreceptor cell lines. We found that the V3 region chimeras failed to replicate in T lymphocyte cell lines but replicated in MDMs and PBLs, albeit at reduced levels compared with R5 laboratory HIV-1 strains. In addition, the V3 region chimeras were able to infect the HOS-CD4(+)CCR5(+) cell line, suggesting CCR5 coreceptor utilization. Moreover, the V3 region chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium-inducing (NSI) phenotypes. In conclusion, the HIV-1 minor genotypes of infected mothers with macrophage-tropic and NSI or R5 phenotypes are transmitted to their infants and are initially maintained with the same properties.

title: Evaluation of Genetic Diversity of Human Immunodeficiency Virus Type 1 nef Gene Associated with Vertical Transmission
abstract: The nef gene is conserved among members of human and simian immunodeficiency viruses and may play an important role in viral pathogenesis. To determine the evolutionary dynamics and conservation of functionality of the human immunodeficiency virus type 1 (HIV-1) nef gene during maternal-fetal transmission, we analyzed nef sequences from seven mother-infant pairs following perinatal transmission, including a mother with infected twin infants. The nef open reading frame was maintained in mother-infant isolates with a frequency of 86.2% following vertical transmission. While there was a low degree of viral heterogeneity and estimates of genetic diversity and high population growth rates of nef sequences from mother-infant isolates, the infants’ nef sequences were slightly higher with respect to these parameters compared with the mothers’ sequences. Both the mothers’ and infants’ nef sequences were under positive selection pressure, as determined by a new method of Nielsen and Yang [Genetics 148:929–936;1998]. Based on genetic distance and phylogenetic parameters, the epidemiologically linked nef sequences from mother-infant pairs were closer to each other compared with epidemiologically unlinked sequences from individuals. The functional domains essential for Nef activity, including membrane binding, CD4 and MHC-I downmodulation, T cell activation and interaction with factors of the cellular protein trafficking machinery, were conserved in most of the sequences from mother-infant pairs. The maintenance of intact nef open reading frames with conserved functional domains and a low degree of genetic variability following vertical transmission supports the notion that nef plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.

title: Characterization of HIV type 1 tat sequences associated with perinatal transmission.
abstract: Human immunodeficiency virus type 1 (HIV-1) tat exon I sequences were analyzed from six mother-infant pairs after perinatal transmission. The tat open reading frame was maintained in 140 of the 154 clones analyzed, with a 90.9% frequency of intact tat open reading frames. In addition, a low degree of heterogeneity was observed in tat sequences within mothers, within infants, and between epidemiologically linked mother-infant pairs. However, the distances of tat sequences between epidemiologically unlinked individuals were greater than in epidemiologically linked mother-infant pairs. The infant sequences showed amino acid sequence patterns similar to those present in their respective mothers. The functional domains required for Tat function, including amino-terminal, cysteine-rich, core and basic regions, which constitute domains for activation and RNA binding, were highly conserved in most of the sequences. Phylogenetic analysis of 154 mother-infant tat sequences showed that they formed distinct clusters for each mother-infant pair and grouped with subtype B sequence. These findings suggest that an intact and functional tat gene is conserved in HIV-1 mother-infant isolates that are involved in perinatal transmission.

title: Conservation of an Intact vif Gene of Human Immunodeficiency Virus Type 1 during Maternal-Fetal Transmission
abstract: The human immunodeficiency virus type 1 (HIV-1) vifgene is conserved among most lentiviruses, suggesting thatvif is important for natural infection. To determine whether an intact vif gene is positively selected during mother-to-infant transmission, we analyzed vif sequences from five infected mother-infant pairs following perinatal transmission. The coding potential of the vif open reading frame directly derived from uncultured peripheral blood mononuclear cell DNA was maintained in most of the 78,912 bp sequenced. We found that 123 of the 137 clones analyzed showed an 89.8% frequency of intact vif open reading frames. There was a low degree of heterogeneity of vif genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vif sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Furthermore, the epidemiologically linked mother-infant pairvif sequences displayed similar patterns that were not seen in vif sequences from epidemiologically unlinked individuals. The functional domains, including the two cysteines at positions 114 and 133, a serine phosphorylation site at position 144, and the C-terminal basic amino acids essential for vifprotein function, were highly conserved in most of the sequences. Phylogenetic analyses of 137 mother-infant pair vifsequences and 187 other available vif sequences from HIV-1 databases revealed distinct clusters for vif sequences from each mother-infant pair and for other vif sequences. Taken together, these findings suggest that vif plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.

title: Maintenance of an Intact Human Immunodeficiency Virus Type 1 vpr Gene following Mother-to-Infant Transmission
abstract: The vpr sequences from six human immunodeficiency virus type 1 (HIV-1)-infected mother-infant pairs following perinatal transmission were analyzed. We found that 153 of the 166 clones analyzed from uncultured peripheral blood mononuclear cell DNA samples showed a 92.17% frequency of intact vpr open reading frames. There was a low degree of heterogeneity of vprgenes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vprsequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Moreover, the infants’ sequences displayed patterns similar to those seen in their mothers. The functional domains essential for Vpr activity, including virion incorporation, nuclear import, and cell cycle arrest and differentiation were highly conserved in most of the sequences. Phylogenetic analyses of 166 mother-infant pairs and 195 other available vpr sequences from HIV databases formed distinct clusters for each mother-infant pair and for other vprsequences and grouped the six mother-infant pairs’ sequences with subtype B sequences. A high degree of conservation of intact and functional vpr supports the notion that vprplays an important role in HIV-1 infection and replication in mother-infant isolates that are involved in perinatal transmission.

title: Molecular characterization of HIV type 1 vpu genes from mothers and infants after perinatal transmission.
abstract: We compared 162 vpu sequences of human immunodeficiency virus type 1 (HIV-1) in peripheral blood mononuclear cell DNA of 6 infected mother-infant pairs after perinatal transmission, and found a 90.12% frequency of intact vpu open reading frames. The heterogeneity of vpu genes between epidemiologically linked mother-infant pairs was lower compared with epidemiologically unlinked individuals. However, the variability of vpu genes was higher than that seen for other HIV-1 genes, including vif, vpr, tat, and gag p17 from the same mother-infant pairs. Moreover, the infants' sequences displayed patterns similar to those seen in their mothers. The functional domains essential for Vpu activity, including efficient release of virus particles from infected cells and CD4 degradation, were conserved in most of the sequences. In a phylogenetic analysis, the 162 sequences from 6 mother-infant pairs formed distinct clusters for each mother-infant pair sequences and grouped with subtype B sequences. These data support the importance of vpu in HIV-1 replication of mother-infant isolates that are involved in perinatal transmission.

title: Low conservation of functional domains of HIV type 1 vif and vpr genes in infected mothers correlates with lack of vertical transmission.
abstract: Human immunodeficiency virus type 1 (HIV-1) vif and vpr sequences were analyzed from four nontransmitting mothers (infected mothers who failed to transmit HIV-1 to their infants mainly in the absence of anti-retroviral therapy), including a mother with multiple deliveries, and compared with the vif and vpr sequences of five and six previously analyzed transmitting mothers, respectively. In contrast to a high functional conservation of vif and vpr genes in transmitting mother isolates, we found that there was a low degree of conservation of functional domains of these genes in nontransmitting (NT) mother isolates. For vif sequences, NT-2 contained stop codons and no initiation codons, whereas NT-1 sequences carried a substitution of a highly conserved tyrosine to histidine at position 30. In addition, NT-3 and NT-4 sequences contained additional substitutions, including asparagine at position 22, lysine at position 77 and histidine at position 110, that were absent in transmitting mother and consensus subtype B sequences. Similarly, the vpr sequences of NT-2 contained stop codons and no initiation codons, NT-4 contained a substitution of serine in place of alanine at position 30, some NT-1 sequences substituted arginine in place of glycine at position 75, and NT-3 sequences presented a deletion in the C terminus that was absent in transmitting mother and consensus subtype B sequences and is essential for Vpr function. Furthermore, vif and vpr sequences of nontransmitting mothers were less heterogeneous compared with transmitting mother sequences. In conclusion, a low degree of conservation of functional domains and heterogeneity of HIV-1 vif and vpr genes in these infected mothers correlates with lack of vertical transmission.

title: Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity
abstract: Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.

title: Assembly of Retrovirus Capsid-Nucleocapsid Proteins in the Presence of Membranes or RNA
abstract: Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

title: Differing Roles of the N- and C-terminal Zinc Fingers in Human Immunodeficiency Virus Nucleocapsid Protein-enhanced Nucleic Acid Annealing*
abstract: The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing.

title: HIV-1 gag proteins: diverse functions in the virus life cycle.
abstract: The Gag proteins of HIV-1, like those of other retroviruses, are necessary and sufficient for the assembly of virus-like particles. The roles played by HIV-1 Gag proteins during the life cycle are numerous and complex, involving not only assembly but also virion maturation after particle release and early postentry steps in virus replication. As the individual Gag domains carry out their diverse functions, they must engage in interactions with themselves, other Gag proteins, other viral proteins, lipid, nucleic acid (DNA and RNA), and host cell proteins. This review briefly summarizes our current understanding of how HIV-1 Gag proteins function in the virus life cycle.

title: DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection.
abstract: The nucleocapsid (NC) protein NCp7 of the immunodeficiency virus type 1 is a small basic protein with two zinc finger motifs. NCp7 has key roles in virus replication and structure, which rely on its interactions with nucleic acids. Although most interactions involve RNAs, binding to the viral DNA is thought to be of importance to achieve protection of the DNA against cellular nucleases and its integration into the host genome. We investigated the interaction of NCp7 with plasmid DNA as a model system. The fluorescence probe YOYO-1 was used as the reporter. Binding of NCp7 to DNA caused DNA condensation, as inferred from the dramatic decrease in YOYO-1 fluorescence. Efficient condensation of DNA required the full length NCp7 with the zinc fingers. The fingerless peptide was less efficient in condensing DNA. Binding of both these NC peptides led to freezing of the segmental dynamics of DNA as revealed by anisotropy decay kinetics of YOYO-1. The truncated peptide NC(12-55) which retains the zinc fingers did not lead to DNA condensation despite its ability to bind and partially freeze the segmental motion of DNA. We propose that the histone-like property of NCp7 leading to DNA condensation contributes to viral DNA stability, in vivo.

title: The Human Immunodeficiency Virus Type 1 Gag Polyprotein Has Nucleic Acid Chaperone Activity: Possible Role in Dimerization of Genomic RNA and Placement of tRNA on the Primer Binding Site
abstract: The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.

title: Inhibition of multiple phases of human immunodeficiency virus type 1 replication by a dithiane compound that attacks the conserved zinc fingers of retroviral nucleocapsid proteins
abstract: The human immunodeficiency virus type 1 (HIV-1) nucleocapsid p7 protein contains two retrovirus-type zinc finger domains that are required for multiple phases of viral replication. Chelating residues (three Cys residues and one His residue) of the domains are absolutely conserved among all strains of HIV-1 and other retroviruses, and mutations in these residues in noninfectious virions. These properties establish the zinc finger domains as logical targets for antiviral chemotherapy. Selected dithiobis benzamide (R-SS-R) compounds were previously found to inhibit HIV-1 replication by mediating an electrophilic attack on the zinc fingers. Unfortunately, reaction of these disulfide-based benzamides with reducing agents yields two monomeric structures (two R-SH structures) that can dissociated and no longer react with the zinc fingers, suggesting that in vivo reduction would inactivate the compounds. Through an extensive drug discovery program of the National Cancer Institute, a nondissociable tethered dithiane compound (1,2-dithiane-4,5-diol, 1,1-dioxide, cis; NSC 624151) has been identified. This compound specifically attacks the retroviral zinc fingers, but not other antiviral targets. The lead compound demonstrated broad antiretroviral activity, ranging from field isolates and drug-resistant strains of HIV-1 to HIV-2 and simian immunodeficiency virus. The compound directly inactivated HIV-1 virions and blocked production of infectious virus from cells harboring integrated proviral DNA. NSC 624151 provides a scaffold from which medicinal chemists can develop novel compounds for the therapeutic treatment of HIV infection.

title: Destabilization of the HIV-1 complementary sequence of TAR by the nucleocapsid protein through activation of conformational fluctuations.
abstract: The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3' terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12-55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12-55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR-TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.

title: Human Cellular Nucleic Acid-Binding Protein Zn2+ Fingers Support Replication of Human Immunodeficiency Virus Type 1 When They Are Substituted in the Nucleocapsid Protein
abstract: A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn2+ fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn2+ fingers. The sequence of the NH2-terminal NC Zn2+ finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn2+ finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn2+ fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn2+ finger compared to mutant and wild-type Zn2+ fingers in NC that support replication. The present study shows that six of seven of the Zn2+ fingers from the CNBP protein can be used as substitutes for the Zn2+ finger in the NH2-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn2+ finger mutants.

title: A complementary single-stranded docking site is required for enhancement of strand exchange by human immunodeficiency virus nucleocapsid protein on substrates that model viral recombination.
abstract: Enhancement of strand exchange by nucleocapsid protein (NC) is proposed to occur during retroviral recombination. The mechanism was examined using an RNA (donor)-DNA hybrid that mimicked a retrovirus replication intermediate. This consisted of a 25 base pair hybrid region flanked on each side by single-stranded RNA or DNA. A second set of acceptor RNAs that could bind to the 25-base hybrid region and to various lengths of additional bases on the DNA was used to displace the donor by hybridizing with the DNA. Displacement required a complementary single-stranded DNA region outside the donor-DNA 25-nucleotide hybrid region. NC enhanced displacement slightly when the acceptor could bind 10 nucleotides and significantly when binding 22 or more nucleotides in the single-stranded region. Two mutated acceptors that bound over 47 total nucleotides on the DNA (22 in the single-stranded region plus 25 in the hybrid region) were constructed. One had three mismatches in the hybrid region; the other, three in the single-stranded region and one in the hybrid region. Each acceptor bound the DNA with approximately equal thermodynamic stability, yet NC stimulated exchange with the former and actually inhibited with the latter. This emphasized the importance of the single-stranded region in NC stimulation. The results support a mechanism where NC enhances the docking of the acceptor to the single-stranded region and then the acceptor "zippers" through the hybrid and displaces the donor. Results with the mutated acceptors indicate that NC may actually inhibit strand exchange between genomes in nonhomologous regions.

title: Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism.
abstract: Publisher Summary This chapter focuses on recent biochemical and biophysical studies that examine the nucleic acid chaperone function of HIV‐1 nucleocapsid protein (NC) and its critical role in facilitating specific and efficient reverse transcription. This chapter also describes the effect of NC on individual steps in viral DNA synthesis. This chapter also summarizes what is known about NC structure, NC nucleic acid binding properties, and the contribution of the zinc fingers to chaperone activity. In addition, this chapter also discusses new evidence that provides a model to explain the mechanism of NC's nucleic acid chaperone activity at the molecular level. Characterization of the mechanism of NC's chaperone activity in molecular terms has been invaluable for understanding NC's effect on specific steps in reverse transcription. NC binds nucleic acids noncooperatively and does not rely on protein– protein interactions to drive aggregation and annealing. Instead, NC‐induced nucleic acid aggregation appears to be facilitated by simple polyelectrolyte attraction, similar to that observed for many multivalent cations.

title: G-quartets direct assembly of HIV-1 nucleocapsid protein along single-stranded DNA.
abstract: The d(TTGGGGGGTACAGTGCA) sequence, derived from the human immunodeficiency virus type 1 (HIV-1) central DNA flap, can form in vitro an intermolecular parallel DNA quadruplex. This work demonstrates that the HIV-1 nucleocapsid protein (NCp) exhibits a high affinity (10(8) M(-1)) for this quadruplex. This interaction is predominantly hydrophobic, maintained by a stabilization between G-quartet planes and the C-terminal zinc finger of the protein. It also requires 5 nt long tails flanking the quartets plus both the second zinc-finger and the N-terminal domain of NCp. The initial binding nucleates an ordered arrangement of consecutive NCp along the four single-stranded tails. Such a process requires the N-terminal zinc finger, and was found to occur for DNA site sizes shorter than usual in a sequence-dependent manner. Concurrently, NCp binding is efficient on a G'2 quadruplex also derived from the HIV-1 central DNA flap. Apart from their implication within the DNA flap, these data lead to a model for the nucleic acid architecture within the viral nucleocapsid, where adjacent single-stranded tails and NCp promote a compact assembly of NCp and nucleic acid growing from stably and primary bound NCp.

title: Identification of the 15FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion
abstract: The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx)4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu44 and Phe45 in the (Lxx)4 domain or deletion of the whole (Lxx)4 domain (amino acid #35–52 of the Gag p6 domain) did not affect Vpr virion packaging. Vpr virion packaging was normal when amino acid #1–23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe15, Arg16 and Phe17 in the context of amino acid #1–23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe15 and Phe17 also abolished Vpr virion packaging, whereas Ala substitution for Arg16 had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The 15FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores.

title: AIP1/ALIX Is a Binding Partner for HIV-1 p6 and EIAV p9 Functioning in Virus Budding
abstract: HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.

title: A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr
abstract: The vpr gene product of human immunodeficiency virus type (HIV-1) is a virion-associated regulatory protein. A transferable virion association motif for Vpr is located in the p6 domain of the HIV-1 Gag polyprotein. To map the sequences in p6 that are involved in Vpr incorporation, we analyzed the ability of mutant forms of p6 to direct the incorporation of Vpr into chimeric viral particles. Our results show that the determinants which govern Vpr incorporation are largely confined to a C-terminal region of the p6 domain. Within this region, three hydrophobic residues in a highly conserved sequence motif (L-X-S-L-F-G) are absolutely required. Remarkably, the transfer of the conserved motif and of a single flanking residue to a heterologous Gag polyprotein was sufficient to transfer the ability to incorporate Vpr at moderate levels. The transfer of residues 32 to 46 of p6 led to Vpr incorporation levels that were comparable to those obtained with full-length HIV-1 Gag protein, indicating that this region contains essentially all the information required for efficient Vpr incorporation.

title: Incorporation of Vpr into Human Immunodeficiency Virus Type 1 Requires a Direct Interaction with the p6 Domain of the p55 Gag Precursor*
abstract: The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55 gag ), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55 gag corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein istrans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55 gag using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55 gag can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6 gag completely abolish the interaction between Vpr and the Pr55 gag and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55 gag . Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal α-helical structure is essential for the Vpr-Pr55 gag interaction. Vpr and the Pr55 gag demonstrate a strong interaction in vitroas salt concentrations as high as 900 mm could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vprtrans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6 gag . The development of Pr55 gag -Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.

title: Tsg101 Control of Human Immunodeficiency Virus Type 1 Gag Trafficking and Release
abstract: The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55gag, contains a proline-rich motif (PTAP) called the “late domain” in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.

title: Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release.
abstract: Mutations in sequences at the C terminus of the capsid precursor protein of human immunodeficiency virus type 1 that affect the viral p6 protein prevent release of budded virus particles from the cell surface. The experiments reported here define an important step in the life cycle of the virus, the release of the budded particle from a tether that binds the assembled particle to the cell surface. Inhibition of the release of the viral capsid proteins by interferon alpha indicates that this step of virus maturation may be sensitive to inhibition by antiviral drugs.

title: Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function
abstract: Efficient budding of HIV-1 from the plasma membrane of infected cells requires the function of a 6-kDa protein known as p6. A highly conserved Pro-Thr-Ala-Pro (PTAP) motif (the “late” or “L” domain), is critical for the virus-budding activity of p6. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N terminus a domain highly related to ubiquitin-conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle assembly and release. We observed that this domain (referred to as TSG-5′) potently inhibits virus production. Examination of cells coexpressing HIV-1 Gag and TSG-5′ by electron microscopy reveals a defect in virus budding reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5′ depends on an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5′. Furthermore, assembly and release of murine leukemia virus and Mason–Pfizer monkey virus are insensitive to TSG-5′. TSG-5′ is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5′ incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and indicate that TSG101 derivatives can act as potent and specific inhibitors of HIV-1 replication by blocking virus budding.

title: Tsg101 and the Vacuolar Protein Sorting Pathway Are Essential for HIV-1 Budding
abstract: Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

title: The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner
abstract: The p6 domain of human immunodeficiency virus type 1 (HIV-1) is located at the C terminus of the Gag precursor protein Pr55Gag. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.

title: Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins
abstract: The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein–protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.

title: Late Assembly Domain Function Can Exhibit Context Dependence and Involves Ubiquitin Residues Implicated in Endocytosis
abstract: Retroviral Gag polyproteins contain regions that promote the separation of virus particles from the plasma membrane and from each other. These Gag regions are often referred to as late assembly (L) domains. The L domain of human immunodeficiency virus type 1 (HIV-1) is in the C-terminal p6 gag domain and harbors an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses are in the N-terminal half of the Gag precursor and have a PPXY core motif. We recently observed that L domains induce the ubiquitination of a minimal HIV-1 Gag construct and that point mutations which abolish L domain activity prevent Gag ubiquitination. In that study, a peptide from the Ebola virus L domain with overlapping P(T/S)APP and PPXY motifs showed exceptional activity in promoting Gag ubiquitination and the release of virus-like particles. We now show that a substitution which disrupts the PPXY motif but leaves the P(T/S)APP motif intact abolishes L domain activity in the minimal Gag context, but not in the context of a near full-length HIV-1 Gag precursor. Our results reveal that the P(T/S)APP motif does not function autonomously and indicate that the HIV-1 nucleocapsid-p1 region, which is proximal to p6 gag , can cooperate with the conserved L domain core motif. We have also examined the effects of ubiquitin mutants on virus-like particle production, and the results indicate that residues required for the endocytosis function of ubiquitin are also involved in virus budding.

title: Biochemical Analyses of the Interactions between Human Immunodeficiency Virus Type 1 Vpr and p6Gag
abstract: The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (∼200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6Gag domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr1–97) or the amino-terminal 71 amino acids (Vpr1–71) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of ∼75 μM. Gel filtration and cross-linking analyses further revealed that Vpr1–71 self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.

title: A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles
abstract: Incorporation of Vpr into human immunodeficiency virus type 1 (HIV-1) virions is mediated by the Gag protein, independently of other viral components. We have coexpressed Vpr and Gag constructs in a vaccinia virus expression system in order to map the region of Gag involved in Vpr packaging. Deletion of the carboxyl-terminal p6 region of Gag impaired the ability of Gag to package Vpr. To confirm the role of p6 in Vpr packaging, Rous sarcoma virus (RSV)-HIV chimeras containing HIV-1 p6 were constructed. Although RSV Gag does not package Vpr into virus particles, a chimera containing HIV-1 p6 is sufficient for Vpr incorporation. To map the region of p6 involved in Vpr packaging, a series of p6 point mutations and deletion mutations was analyzed. Mutations in the N-terminal p6 proline-rich domain, for which preliminary evidence shows a marked decrease in virion incorporated RNA, did not affect Vpr incorporation. Deletion of residues 1 to 31 of HIV-1 p6 did not affect Vpr packaging, but residues 35 to 47, including an (LXX)4 domain, were required for Vpr incorporation into virus particles.

title: MODELTEST: testing the model of DNA substitution
abstract: The program MODELTEST uses log likelihood scores to establish the model of DNA evolution that best fits the data.


AVAILABILITY
The MODELTEST package, including the source code and some documentation is available at http://bioag.byu. edu/zoology/crandall_lab/modeltest.html.

title: Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene.
abstract: Several codon-based models for the evolution of protein-coding DNA sequences are developed that account for varying selection intensity among amino acid sites. The "neutral model" assumes two categories of sites at which amino acid replacements are either neutral or deleterious. The "positive-selection model" assumes an additional category of positively selected sites at which nonsynonymous substitutions occur at a higher rate than synonymous ones. This model is also used to identify target sites for positive selection. The models are applied to a data set of the V3 region of the HIV-1 envelope gene, sequenced at different years after the infection of one patient. The results provide strong support for variable selection intensity among amino acid sites The neutral model is rejected in favor of the positive-selection model, indicating the operation of positive selection in the region. Positively selected sites are found in both the V3 region and the flanking regions.

title: Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions.
abstract: Two simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions are presented. Although they give no weights to different types of codon substitutions, these methods give essentially the same results as those obtained by Miyata and Yasunaga's and by Li et al.'s methods. Computer simulation indicates that estimates of synonymous substitutions obtained by the two methods are quite accurate unless the number of nucleotide substitutions per site is very large. It is shown that all available methods tend to give an underestimate of the number of nonsynonymous substitutions when the number is large.

title: Conservation of functional domains and limited heterogeneity of HIV-1 reverse transcriptase gene following vertical transmission
abstract: BackgroundThe reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the life cycle of the virus by converting the single stranded RNA genome into double stranded DNA that integrates into the host chromosome. In addition, RT is also responsible for the generation of mutations throughout the viral genome, including in its own sequences and is thus responsible for the generation of quasi-species in HIV-1-infected individuals. We therefore characterized the molecular properties of RT, including the conservation of functional motifs, degree of genetic diversity, and evolutionary dynamics from five mother-infant pairs following vertical transmission.ResultsThe RT open reading frame was maintained with a frequency of 87.2% in five mother-infant pairs' sequences following vertical transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' sequences. Both mothers and infants RT sequences were under positive selection pressure, as determined by the ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 132 mother-infant RT sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked mother-infant pairs. The functional domains of RT which are responsible for reverse transcription, DNA polymerization and RNase H activity were mostly conserved in the RT sequences analyzed in this study. Specifically, the active sites and domains required for primer binding, template binding, primer and template positioning and nucleotide recruitment were conserved in all mother-infant pairs' sequences.ConclusionThe maintenance of an intact RT open reading frame, conservation of functional domains for RT activity, preservation of several amino acid motifs in epidemiologically linked mother-infant pairs, and a low degree of genetic variability following vertical transmission is consistent with an indispensable role of RT in HIV-1 replication in infected mother-infant pairs.

title: Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein
abstract: The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprotein correlated well with the effects of the mutations on particle-associated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.

title: Resistance to Nucleoside Analog Reverse Transcriptase Inhibitors Mediated by Human Immunodeficiency Virus Type 1 p6 Protein
abstract: Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within thepol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6 gag -p6 pol region of HIV-1, placed immediately upstream ofpol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6Gag), or by changes in activation of the viral protease (p6Pol). Duplication of the proline-rich p6Gag PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naı̈ve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6Pol), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

title: Important contribution of p15 Gag-specific responses to the total Gag-specific CTL responses
abstract: Objectives HIV-1 p15 Gag and its protease cleavage products, NCp7 and p6, are believed to play a major role in viral infectivity and assembly during the early and late stages of the retroviral life cycle. However, the extent to which p15 Gag is targeted by the host immune system in natural infection as well as precise cytotoxic T lymphocyte (CTL) epitopes within this protein remains to be defined.  In this study, 57 HIV-1 infected individuals and 10 HIV-1 negative controls were screened for CD8 and CD4 T-cell responses using overlapping peptides spanning the entire p15 Gag protein as well as the p17 Gag and p24 Gag proteins. Peptide-specific interferon-γ production was measured by Elispot assay and flow-based intracellular cytokine quantification, and cytotoxic activity was confirmed after isolation of peptide-specific CD8 T-cell lines. Results CD8 T lymphocytes specific to p15 Gag were found in 46% (26/57) of HIV-1 infected individuals studied and contributed on average 17% (range, 0–100%) to the total Gag-specific T-cell responses. Responses were clustered within three immunodominant regions of p15 Gag, mapping to important functional sites. These studies also include the description of the first three optimally defined CTL epitopes within p15 Gag.  These results indicate that p15 Gag is frequently recognized by HIV-1-specific CD8 T cells in HIV-1 infection and will be important in the comprehensive assessments of CTL responses in infected persons, as well as the design and testing of future HIV-1 vaccines and immunotherapeutic interventions.

title: Frequent Detection of Escape from Cytotoxic T-Lymphocyte Recognition in Perinatal Human Immunodeficiency Virus (HIV) Type 1 Transmission: the Ariel Project for the Prevention of Transmission of HIV from Mother to Infant
abstract: Host immunologic factors, including human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL), are thought to contribute to the control of HIV type 1 (HIV-1) replication and thus delay disease progression in infected individuals. Host immunologic factors are also likely to influence perinatal transmission of HIV-1 from infected mother to infant. In this study, the potential role of CTL in modulating HIV-1 transmission from mother to infant was examined in 11 HIV-1-infected mothers, 3 of whom transmitted virus to their offspring. Frequencies of HIV-1-specific human leukocyte antigen class I-restricted CTL responses and viral epitope amino acid sequence variation were determined in the mothers and their infected infants. Maternal HIV-1-specific CTL clones were derived from each of the HIV-1-infected pregnant women. Amino acid substitutions within the targeted CTL epitopes were more frequently identified in transmitting mothers than in nontransmitting mothers, and immune escape from CTL recognition was detected in all three transmitting mothers but in only one of eight nontransmitting mothers. The majority of viral sequences obtained from the HIV-1-infected infant blood samples were susceptible to maternal CTL. These findings demonstrate that epitope amino acid sequence variation and escape from CTL recognition occur more frequently in mothers that transmit HIV-1 to their infants than in those who do not. However, the transmitted virus can be a CTL susceptible form, suggesting inadequate in vivo immune control.

title: Genetic characterization of HIV type 1 gag p17 matrix genes in isolates from infected mothers lacking perinatal transmission.
abstract: The gag p17 matrix sequences of human immunodeficiency virus type 1 (HIV-1) were analyzed from three nontransmitting mothers (mothers who failed to transmit HIV-1 to their infants in the absence of antiretroviral therapy), including multiple deliveries in the case of mother 3. There was a low degree of heterogeneity of gag p17 matrix sequences in nontransmitting mothers compared with our previously analyzed mother-infant pairs' sequences. Whereas most of the functional domains essential for p17 matrix function were generally conserved, the polymerization site was less conserved. Several amino acid motifs, including KIEEEQN (positions 103-109) at the major antibody-binding site, were variable and the C-terminal 6-mer QVSQNY, a lysine or glutamine at position 15, an alanine at position 54, a lysine at position 76, a valine at position 104, and an aspartic acid at positions 102 and 121 were conserved in nontransmitting mothers' sequences compared with transmitting mothers' sequences. Phylogenetic analyses of 82 p17 matrix sequences revealed distinct clusters for each nontransmitting mother. Some of these motifs in gag p17 matrix sequences that are present in nontransmitting mothers and absent in transmitting mothers could be used as new targets for the development of preventive strategies for perinatal transmission.

title: Characterization of mother-infant HIV type 1 gag p17 sequences associated with perinatal transmission.
abstract: The gag p17 matrix sequences of human immunodeficiency virus type 1 (HIV-1) from seven infected mother-infant pairs were analyzed after perinatal transmission. The p17 matrix open reading frame was maintained in 143 of the 166 clones analyzed (86.2% frequency of intact p17 open reading frames). The functional domains essential for p17 matrix function in HIV-1 replication, including targeting of Gag to the plasma membrane, virus assembly and release, envelope glycoprotein incorporation into virus particle, virus entry, and localization of the virus preintegration complex to the nucleus of nondividing cells, were highly conserved in most of the sequences. In addition, examination of the three-dimensional structure of the p17 matrix protein in mother-infant isolates showed a high degree of conservation of amino acids required for correct folding and biological activity. Several amino acid motifs common to most of the mother-infant pairs sequences, including pair-specific signature sequences, were observed. There was a low degree of heterogeneity of gag p17 sequences within mothers, within infants, and between mother-infant pairs, but the distances were greater between epidemiologically unlinked individuals. Phylogenetic analyses of 166 mother-infant pairs and 181 other p17 sequences available from HIV-1 databases revealed distinct clusters for each mother-infant pair and for other p17 sequences. In conclusion, these findings indicate that an intact and functional gag p17 matrix is maintained during maternal-fetal transmission and that several motifs in p17 may be associated with perinatal transmission.

title: Attenuation of HIV-1 Replication in Primary Human Cells with a Designed Zinc Finger Transcription Factor*
abstract: Small molecule inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been extremely successful but are associated with a myriad of undesirable effects and require lifelong daily dosing. In this study we explore an alternative approach, that of inducing intracellular immunity using designed, zinc finger-based transcription factors. Three transcriptional repression proteins were engineered to bind sites in the HIV-1 promoter that were expected to be both accessible in chromatin structure and highly conserved in sequence structure among the various HIV-1 subgroups. Transient transfection assays identified one factor, KRAB-HLTR3, as being able to achieve 100-fold repression of an HIV-1 promoter. Specificity of repression was demonstrated by the lack of repression of other promoters. This factor was further shown to repress the replication of several HIV-1 viral strains 10- to 100-fold in T-cell lines and primary human peripheral blood mononuclear cells. Repression was observed for at least 18 days with no significant cytotoxicity. Stable T-cell lines expressing the factor also do not show obvious signs of cytotoxicity. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.

title: Enhancement of Primary and Secondary Cellular Immune Responses against Human Immunodeficiency Virus Type 1 Gag by Using DNA Expression Vectors That Target Gag Antigen to the Secretory Pathway
abstract: In this study, we have investigated the influence of antigen targeting after DNA vaccination upon the induction of cellular immune responses against human immunodeficiency virus type 1 (HIV-1) Gag. In addition to the standard version of HIV-1 Gag, we constructed Gag expression vectors that encode a secreted (Sc-Gag) and a cytoplasmic (Cy-Gag) Gag molecule. Although all three HIV-1 Gag expression vectors induced detectable humoral and cellular immune responses, after intramuscular injection the DNA vector encoding the Sc-Gag generated the highest primary cytotoxic T-lymphocyte (CTL) and T-helper responses. Mice immunized with one of the HIV-1 Gag DNA vectors (but not with the control vector pcDNA3.1) developed a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag, and this response persisted for 125 days. The magnitude of the protection correlated with the levels of Gag-specific ex vivo CTL activity and the number of CD8+ T cells producing gamma interferon. The DNA vector encoding the Sc-Gag induced higher levels of protection and greater secondary CTL responses than did the DNA vector encoding Cy-Gag.

title: Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within thegag-pol Transframe Domain
abstract: Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3′ end of the HIV-1 gag-poltransframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6Gag protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNAPR2. A disrupted translation of gag-pol mRNA induced at the PNAPR2-annealing site resulted in a decreased synthesis of Pr160Gag-Pol polyprotein, hence the viral protease, a predominant expression of Pr55Gag devoid of a fully functional p6Gag protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNAPR2abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.

title: Prevalence of Drug‐Resistant Human Immunodeficiency Virus Type 1 in Therapy‐Naive Patients and Usefulness of Genotype Testing
abstract: In the present study, we performed genotypic drug‐resistance testing in 116 therapy‐naive human immunodeficiency virus type 1 (HIV‐1)‐infected patients between 1999 and 2002 at Nagoya National Hospital, Japan. The prevalence of drug‐resistant HIV‐1 with one or more major mutations significantly increased from 5.3% (4/75) in 1999–2001 to 17.1% (7/41) in 2002 (P=0.05), suggesting the spread of drug‐resistant HIV‐1. We identified a patient who possessed a protease (PR) inhibitor‐resistant HIV‐1 with a major mutation consisting of L90M before the initiation of therapy. The patient was administered zidovudine, lamivudine, and efavirenz as highly active antiretroviral therapy (HAART), as PR inhibitors were excluded based on the result of the drug‐resistance testing. The treatment succeeded in strongly suppressing the proliferation of drug‐resistant HIV‐1 and concomitantly increased CD4 cell counts. Thus, we conclude that drug‐resistance testing prior to the initiation of therapy is important for therapy‐naive patients to devise the optimum therapy regimen for each individual.

title: Human Immunodeficiency Virus-Specific CD8+ T Cells in Human Breast Milk
abstract: Breast-feeding infants of human immunodeficiency virus (HIV)-infected women ingest large amounts of HIV, but most escape infection. While the factors affecting transmission risk are poorly understood, HIV-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in controlling HIV levels in blood. We therefore investigated the ability of breast milk cells (BMC) from HIV-infected women from the United States and Zambia to respond to HIV-1 peptides in a gamma interferon enzyme-linked immunospot assay. All (n = 11) HIV-infected women had responses to pools of Gag peptide (range, 105 to 1,400 spot-forming cells/million; mean = 718), 8 of 11 reacted to Pol, 7 reacted to Nef, and 2 of 5 reacted to Env. Conversely, of four HIV-negative women, none responded to any of the tested HIV peptide pools. Depletion and tetramer staining studies demonstrated that CD8+ T cells mediated these responses, and a chromium-release assay showed that these BMC were capable of lysing target cells in an HIV-specific manner. These data demonstrate the presence of HIV-specific major histocompatibility complex class I-restricted CD8+ CTLs in breast milk. Their presence suggests a role in limiting transmission and provides a rationale for vaccine strategies to enhance these responses.

title: Estimating effective population size and mutation rate from sequence data using Metropolis-Hastings sampling.
abstract: We present a new way to make a maximum likelihood estimate of the parameter 4N mu (effective population size times mutation rate per site, or theta) based on a population sample of molecular sequences. We use a Metropolis-Hastings Markov chain Monte Carlo method to sample genealogies in proportion to the product of their likelihood with respect to the data and their prior probability with respect to a coalescent distribution. A specific value of theta must be chosen to generate the coalescent distribution, but the resulting trees can be used to evaluate the likelihood at other values of theta, generating a likelihood curve. This procedure concentrates sampling on those genealogies that contribute most of the likelihood, allowing estimation of meaningful likelihood curves based on relatively small samples. The method can potentially be extended to cases involving varying population size, recombination, and migration.