Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non-poisson distribution of transmitted variants.

Identifying the specific genetic characteristics of successfully transmitted variants may prove central to the development of effective vaccine and microbicide interventions. Although human immunodeficiency virus transmission is associated with a population bottleneck, the extent to which different factors influence the diversity of transmitted viruses is unclear. We estimate here the number of transmitted variants in 69 heterosexual men and women with primary subtype C infections. From 1,505 env sequences obtained using a single genome amplification approach we show that 78% of infections involved single variant transmission and 22% involved multiple variant transmissions (median of 3). We found evidence for mutations selected for cytotoxic-T-lymphocyte or antibody escape and a high prevalence of recombination in individuals infected with multiple variants representing another potential escape pathway in these individuals. In a combined analysis of 171 subtype B and C transmission events, we found that infection with more than one variant does not follow a Poisson distribution, indicating that transmission of individual virions cannot be seen as independent events, each occurring with low probability. While most transmissions resulted from a single infectious unit, multiple variant transmissions represent a significant fraction of transmission events, suggesting that there may be important mechanistic differences between these groups that are not yet understood.

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1.

Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.

The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.

A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation.

Hepatitis C virus (HCV) initiates translation of its polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of the 341-nucleotide (nt) 5' nontranslated RNA (5'NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which secondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5' border of the HCV IRES and thus may function in viral translation. This prediction was tested by a mutational analysis of putative helical structures that examined the impact of both covariant and noncovariant nucleotide substitutions on IRES activity in vivo and in vitro. Results of these experiments provide support for predicted base pair interactions between nt 44 to 52 and 111 to 118 and between nt 65 to 70 and 97 to 102 of the HCV 5'NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted in reciprocal changes in V1 nuclease cleavage patterns within the opposing strand of the putative helix, consistent with the predicted base pair interactions. IRES activity was highly dependent on maintenance of the stem-loop II structure but relatively tolerant of covariant nucleotide substitutions within predicted helical segments. Sequence alignments suggested that the deduced domain II structure is conserved within the IRESs of pestiviruses as well as the novel flavivirus GB virus B. Despite marked differences in primary nucleotide sequence within conserved helical segments, the sequences of the intervening single-stranded loop segments are highly conserved in these different viruses. This suggests that these segments of the viral RNA may interact with elements of the host translational machinery that are broadly conserved among different mammalian species.

Identification of an immunodominant antigenic site involving the capsid protein VP3 of hepatitis A virus.

Hepatitis A virus, an hepatotropic picornavirus, is a common cause of acute hepatitis in man for which there is no available vaccine. Competitive binding studies carried out in solid phase suggest that neutralizing monoclonal antibodies to hepatitis A virus recognize a limited number of epitopes on the capsid surface, although the polypeptide locations of these epitopes are not well defined. Neutralization-escape mutants, selected for resistance to monoclonal antibodies, demonstrate broad cross-resistance to other monoclonal antibodies. Sequencing of virion RNA from several of these mutants demonstrated that replacement of aspartic acid residue 70 of capsid protein VP3 (residue 3070) with histidine or alanine confers resistance to neutralization by monoclonal antibody K2-4F2 and prevents binding of this antibody and other antibodies with similar solid-phase competition profiles. These results indicate that residue 3070 contributes to an immunodominant antigenic site. Mutation at residue 102 of VP1 (residue 1102) confers partial resistance against antibody B5-B3 and several other antibodies but does not prevent antibody attachment. Both VP3 and VP1 sites align closely in the linear peptide sequences with sites of neutralization-escape mutations in poliovirus and human rhinovirus, suggesting conservation of structure among these diverse picornaviruses. However, because partial neutralization resistance to several monoclonal antibodies (2D2, 3E1, and B5-B3) was associated with mutation at either residue 3070 or residue 1102, these sites appear more closely related functionally in hepatitis A virus than in these other picornaviruses.

Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA.

Cap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5' nontranslated RNA (5'NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5'NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5'NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5'NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translation in vitro in rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28-69 of the 5'NTR (stem-loop IIa) sharply reduced capsid translation both in vitro and in vivo. A small deletion mutation involving nt 328-334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation both in vitro and in vivo, while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5'NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation.

An infectious cDNA clone of a cytopathic hepatitis A virus: genomic regions associated with rapid replication and cytopathic effect.

Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from cell culture-adapted rr/cpe- HAV. To determine which mutations in one rr/cpe+ virus, HM175/18f, determine enhanced replication in BS-C-1 cells, a series of chimeric viruses was rescued from infectious cDNAs in which HM175/18f genomic segments were placed within the background of a related rr/cpe- virus, HAV/7. Chimeric viruses containing the P2 region of HM175/18f produced replication foci in BS-C-1 cells that were larger than HAV/7, but not as large as HM175/18f virus. Enhanced viral replication required mutations in both 2B and 2C proteins, suggesting that these proteins remain closely associated during replication. Mutations in 5' nontranslated RNA (5'NTR) or P3 proteins had no independent effect, but acted cooperatively with mutations in P2 proteins to enhance replication and render the virus capable of conventional plaque formation. Cytopathic effects correlated with viral replication capacity and were not the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5'NTR, P2, and P3 segments. These results suggest novel interactions between the 5'NTR and P2 proteins during HAV replication and provide useful new infectious cDNA clones.

Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination.

Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype.

Hepatitis C virus core protein induces apoptosis and impairs cell-cycle regulation in stably transformed Chinese hamster ovary cells.

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma. Several lines of evidence suggest that the core protein of HCV may play a role in the development of this cancer. The authors examined regulation of the cell cycle in stable cell lines derived from Chinese hamster ovary (CHO-K1) cells that constitutively expressed one or more of the structural proteins of HCV. In media containing low concentrations of serum (serum starvation), cell lines expressing the core protein showed a significantly lower population of viable cells than noncore-expressing cells. The low viability of the core-expressing cells was a result of the increased population of cells undergoing apoptosis. Interestingly, the cell cycle analysis revealed that the arresting function at G(0) was impaired, and the cell cycle was accelerated in core-expressing cell lines even under serum starvation. Thus, the HCV core protein sensitizes the apoptosis to serum starvation, although it promotes the cell cycle in CHO-K1 cells. To explain these findings, the authors examined the expression of revival apoptosis and cell-cycle-related genes. Expression of the c-myc genes was significantly induced in core-expressing cells in response to serum starvation. Other apoptosis-inducing genes downstream of c-myc, p53, p21WAF1/CIP1 and Bax were significantly highly induced, although there was no induction of Bcl-2, which prevents apoptosis in core-expressing cells. Thus, the HCV core protein induced apoptosis and impaired the regulation of the cell cycle by activating c-myc expression, whereas the p53 and Bax pathways play a role in the induction of apoptosis.

In vivo replication and reversion to wild type of a neutralization-resistant antigenic variant of hepatitis A virus.

Six seronegative owl monkeys were intravenously inoculated with an antigenic variant (S18) of hepatitis A virus that is highly adapted to growth in cell culture and resists neutralization by monoclonal antibodies due to replacement of aspartic acid 70 of capsid protein VP3 with histidine. Each developed hepatitis 22-33 days after inoculation. Virus in feces, serum, and liver was quantified by radioimmunofocus assay. Viremia developed 7-11 days after inoculation, in parallel with fecal shedding of virus, and persisted for a mean of 20.5 days. Although the antigenic variant was recovered from feces or liver of three animals, virus in liver at the time of enzyme elevations was predominantly wild-type antigenic phenotype. Virus was not recovered from liver 96 days after challenge. These studies further define virologic events in hepatitis A and show that in vivo replication of an antigenic variant was restricted compared with that of wild-type virus.

Effects of genital tract inflammation on human immunodeficiency virus type 1 V3 populations in blood and semen.

We have examined cell-free viral populations in the blood plasma and seminal plasma compartments of men infected with subtype C human immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD) clinic for genital tract inflammation in the form of urethritis (n = 43) or a dermatology clinic (controls, n = 14) in Malawi. We have previously shown that the presence of urethritis is associated with an eightfold increase in virus load in the seminal plasma compartment (M. S. Cohen et al., Lancet 349:1868-1873, 1997). The purpose of this study was to determine whether genital tract inflammation and its treatment caused genetic instability in cell-free HIV-1 populations. In a cross-sectional analysis at study entry, three-fourths of the STD and control subjects had multiple V3 populations in their blood while 60% of the STD subjects and 79% of the control subjects had multiple V3 populations in their semen. Overall, one-fourth of all of the subjects showed discordance between results with blood and semen specimens when samples were compared for the presence and absence of subpopulations. When differences in the relative levels of abundance of bands were also taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between STD and control cohorts for these comparisons of the compartments in this cross-sectional analysis at study entry. Longitudinal analysis of the viral populations from two separate clinic visits over 1 to 4 weeks showed that the complexity of each V3 population as measured by Shannon entropy was different in blood and semen at the two time points, indicating that the blood and semen constitute different compartments for HIV-1. The seminal plasma compartment was more dynamic than the blood plasma compartment for the STD subjects who were treated for urethritis, with changes being noted in the presence or absence of V3-HTA bands in the semen of 29% of these subjects but in the blood of only 9% of these subjects. However, the changes were generally small. Overall, our results suggest that 40% of male subjects show discordance between seminal and blood viral populations and that the complexity of each V3 population was different between the two compartments. Both of these results point to the partial independence of the seminal compartment as a viral niche within the body.

Characterization of V3 sequence heterogeneity in subtype C human immunodeficiency virus type 1 isolates from Malawi: underrepresentation of X4 variants.

We have examined the nature of V3 sequence variability among subtype C human immunodeficiency virus type 1 (HIV-1) sequences from plasma-derived viral RNA present in infected men from Malawi. Sequence variability was assessed by direct sequence analysis of the V3 reverse transcription-PCR products, examination of virus populations by a subtype C V3-specific heteroduplex tracking assay (V3-HTA), and selected sequence analysis of molecular clones derived from the PCR products. Sequence variability in V3 among the subtype C viruses was not associated with the presence of basic amino acid substitutions. This observation is in contrast to that for subtype B HIV-1, where sequence variability is associated with such substitutions, and these substitutions are determinants of altered coreceptor usage. Evolutionary variants in subtype C V3 sequences, as defined by the V3-HTA, were not correlated with the CD4 level in the infected person, while such a correlation was found with subtype B V3 sequences. Viruses were isolated from a subset of the subjects; all isolates used CCR5 and not CXCR4 as a coreceptor, and none was able to grow in MT-2 cells, a hallmark of the syncytium-inducing phenotype that is correlated with CXCR4 usage. The overall sequence variability of the subtype C V3 region was no greater than that of the conserved regions of gp120. This limited sequence variability was also a feature of subtype B V3 sequences that do not carry the basic amino acid substitutions associated with altered coreceptor usage. Our results indicate that altered coreceptor usage is rare in subtype C HIV-1 isolates in sub-Saharan Africa and that sequence variability is not a feature of the V3 region of env in the absence of altered coreceptor usage.