Search

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global pork production and has been notorious for its rapid genetic evolution in the field. The nonstructural protein 2 (nsp2) replicase protein represents the fastest evolving region of PRRSV, but the underlying biological significance has remained poorly understood. By deletion mutagenesis, we discovered that the nsp2 hypervariable region plays an important role in controlling the balance of genomic mRNA and a subset of subgenomic mRNAs. More significantly, we revealed an unexpected link of the nsp2 hypervariable region to viral tropism. Specifically, a mutant of the Chinese highly pathogenic PRRSV strain JXwn06 carrying a deletion spanning nsp2 amino acids 323 to 521 (nsp2Δ323--521) in its hypervariable region was found to lose infectivity in primary porcine alveolar macrophages (PAMs), although it could replicate relatively efficiently in the supporting cell line MARC-145. Consequently, this mutant failed to establish an infection in piglets. Further dissection of the viral life cycle revealed that the mutant had a defect (or defects) lying in the steps between virus penetration and negative-stranded RNA synthesis. Taken together, our results reveal novel functions of nsp2 in the PRRSV life cycle and provide important insights into the mechanisms of PRRSV RNA synthesis and cellular tropism. IMPORTANCE The PRRSV nsp2 replicase protein undergoes rapid and broad genetic variations in its middle region in the field, but the underlying significance has remained enigmatic. Here, we demonstrate that the nsp2 hypervariable region not only plays an important regulatory role in maintaining the balance of different viral mRNA species but also regulates PRRSV tropism to primary PAMs. Our results reveal novel functions for PRRSV nsp2 and have important implications for understanding the mechanisms of PRRSV RNA synthesis and cellular tropism. [ABSTRACT FROM AUTHOR]

Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins. Importance The four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.

The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro. We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2= cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells. [ABSTRACT FROM AUTHOR]

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout ( CD300lf F/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV CR6 ) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoV CW3 ) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells ( LysM Cre +) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells ( CD19 Cre ), neutrophils ( Mrp8 Cre ), and dendritic cells ( CD11c Cre ) did not affect MNoV CW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV CW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV CR6 ; that myelomonocytic cells are a major, but not exclusive, target cell of MNoV CW3 ; and that STAT1 signaling restricts the cellular tropism of MNoV CW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis. IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV CR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV CW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV CW3 infection. Mortality associated with the MNoV CW3 strain in Stat1 -/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo ., (Copyright © 2021 American Society for Microbiology.)

Unlabelled: Simian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8(+), CD4(+), and CD19(+) lymphocyte samples and rarely in CD14(+) monocyte and CD56(+) NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/10(5) cells in CD8(+) T lymphocytes, CD4(+) T lymphocytes, and CD19(+) B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4(+) T lymphocytes. SFV DNA levels in CD4(+) T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8(+), CD4(+), and B lymphocytes are major cellular targets of SFV in the blood of infected humans., Importance: Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo. Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4(+) T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-α/β) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-α/β receptor-deficient (IFN-α/βR-/-) mice and primary neuronal cultures. IFN-α/βR-/- mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 ± 0.7 and 3.8 ± 0.5 days after infection with 100 and 10² PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10² PFU showed 62% mortality and a MTD of 11.9 ± 1.9 days. IFN-α/βR-/- mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-α/βR-/- mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-β, either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-α/β controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons. [ABSTRACT FROM AUTHOR]

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVβ3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.

Molecular differences in the envelope glycoproteins of human immunodeficiency virus type 1 and simian immunodeficiency virus (SIV) determine virus infectivity and cellular tropism. To examine how these properties contribute to productive infection in vivo, rhesus macaques were inoculated with strains of single-cycle SIV (scSIV) engineered to express three different envelope glycoproteins with full-length (TM(open)) or truncated (TM(stop)) cytoplasmic tails. The 239 envelope uses CCR5 for infection of memory CD4(+) T cells, the 316 envelope also uses CCR5 but has enhanced infectivity for primary macrophages, and the 155T3 envelope uses CXCR4 for infection of both naive and memory CD4(+) T cells. Separate groups of six rhesus macaques were inoculated intravenously with mixtures of TM(open) and TM(stop) scSIV(mac)239, scSIV(mac)316, and scSIV(mac)155T3. A multiplex real-time PCR assay specific for unique sequence tags engineered into each virus was then used to measure viral loads for each strain independently. Viral loads in plasma peaked on day 4 for each strain and were resolved below the threshold of detection within 4 to 10 weeks. Truncation of the envelope cytoplasmic tail significantly increased the peak of viremia for all three envelope variants and the titer of SIV-specific antibody responses. Although peak viremias were similar for both R5- and X4-tropic viruses, clearance of scSIV(mac)155T3 TM(stop) was significantly delayed relative to the other strains, possibly reflecting the infection of a CXCR4(+) cell population that is less susceptible to the cytopathic effects of virus infection. These studies reveal differences in the peaks and durations of a single round of productive infection that reflect envelope-specific differences in infectivity, chemokine receptor specificity, and cellular tropism.

In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 mum long and 0.5 mum thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.

The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the Staphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

ABSTRACT In the 21st century, several human and swine coronaviruses (CoVs) have emerged suddenly and caused great damage to people's lives and property. The porcine epidemic diarrhea virus (PEDV), leading to enormous economic losses to the pork industry and remains a large challenge. PEDV showed extensive cell tropism, and we cannot ignore the potential risk of cross-species transmission. However, the mechanism of adaptation and cell tropism of PEDV remains largely unknown and in vitro isolation of PEDV remains a huge challenge, which seriously impedes the development of vaccines. In this study, we confirmed that the spike (S) protein determines the adaptability of PEDV to monkey Vero cells and LLC-PK1 porcine cells, and isolated exchange of S1 and S2 subunits of adaptive strains did not make PEDV adapt to cells. Further, we found that the cellular adaptability of rCH/SX/2016-SHNXP depends on S1 and the first half of S2 (S3), and the 803L and 976H of the S2 subunit are critical for rCH/SX/2016-S1HNXP+S3HNXP adaptation to Vero cells. These findings highlight the decisive role of PEDV S protein in cell tropism and the potential role of coronaviruses S protein in cross-species transmissibility. Besides, our work also provides some different insight into finding PEDV receptors and developing PEDV and other coronaviruses vaccines. IMPORTANCE CoVs can spill from an animal reservoir into a naive host to cause diseases in humans or domestic animals. PEDV results in high mortality in piglets, which has caused immense economic losses in the pork industry. Virus isolation is the first step in studying viral pathogenesis and developing effective vaccines. However, the molecular mechanism of PEDV cell tropism is largely unknown, and isolation of endemic PEDV strains remains a major challenge. This study confirmed that the S gene is the decisive gene of PEDV adaptability to monkey Vero cells and porcine LLC-PK1 cells by the PEDV reverse genetics system. Isolated exchange of S1 and S2 of adaptive strains did not make PEDV adapt to cells, and the 803L and 976H of S2 subunit are critical for rCH/SX/2016-S1HNXP+S3HNXP adaptation to Vero cells. These results illustrate the decisive role of PEDV S protein in cell tropism and highlight the potential role of coronaviruses S protein in cross-species transmissibility. Besides, our finding also provides some unique insight into identifying PEDV functional receptors and has guiding significance for developing PEDV and other coronavirus vaccines.

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4(+) T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4(+) lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.

Human herpesvirus 8 (HHV-8) infection has been implicated in the etiology of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), three diseases that frequently develop in immunocompromised, human immunodeficiency virus-positive individuals. One hypothesis that would account for different pathological manifestations of infection by the same virus is that viral genes are differentially expressed in heterogeneous cell types. To test this hypothesis, we analyzed the localization and levels of expression of two viral genes expressed in latent and lytic infections and the viral homologue of interleukin-6 (vIL-6). We show that PEL parallels KS in the pattern of latent and lytic cycle viral gene expression but that the predominant infected cell type is a B cell. We also show that MCD differs from KS not only in the infected cell type (B-cell and T-cell lineage) but also in the pattern of viral gene expression. Only a few cells in the lesion are infected and all of these cells express lytic-cycle genes. Of possibly greater significance is the fact that in a comparison of KS, PEL, and MCD, we found dramatic differences in the levels of expression of vIL-6. Interleukin-6 is a B-cell growth and differentiation factor whose altered expression has been linked to plasma cell abnormalities, as well as myeloid and lymphoid malignancies. Our findings support the hypothesis that HHV-8 plays an important role in the pathogenesis of PEL and MCD, in which vIL-6 acts as an autocrine or paracrine factor in the lymphoproliferative processes common to both.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.

CD4 is the predominant cell membrane protein that binds human immunodeficiency virus type 1 (HIV-1) gp120 and facilitates HIV-1 infection, but other membrane-associated molecules may be involved in determining HIV-1 cellular infection. Our prior work had suggested that CD44, the transmembrane receptor for hyaluronan, might play a role in the infection of mononuclear phagocytes with HIV-1. In the present work, we have used cells of the CD4-positive, CD44-negative human T-lymphoblast cell line Jurkat to study the role of CD44 in HIV-1 infection and tropism. Cells were transfected with cDNA for the standard (S, or hematopoietic) CD44 isoform CD44S or the epithelial isoform CD44E. The resultant lines expressed appropriate CD44S or CD44E mRNA and protein. While the parent Jurkat cells, those transfected with vector alone, and those transfected with CD44E could be productively infected with only the lymphocytotropic strain HIV-1-LAI, cells transfected with CD44S were rendered susceptible to productive infection with the monocytotropic strains HIV-1-BaL and HIV-1-ADA. Also, CD44S-transfected cells displayed higher levels of infection with HIV-1-LAI than did the other transfected Jurkat cells. The transfected cell line cells all had comparable growth rates and expressed similar levels of the membrane antigens CD4, CD7, major histocompatibility complex (MHC) class I, MHC class II, and CD11a, while levels of CD3 were slightly higher in cells transfected with vector alone and in one of the clones transfected with CD44S. Hyaluronan binding was increased in cells transfected with either CD44S or CD44E. Mouse NIH 3T3 fibroblasts transfected with human CD4, human CD44S, or both human CD4 and CD44S displayed the appropriate antigens, but they could not be productively infected with lymphocytotropic or monocytotropic strains of HIV-1. The results indicate that in human leukocytes, CD44S is an important determinant of HIV-1 productive infection and may be involved in viral cellular tropism.

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.

Human immunodeficiency viruses (HIV) isolated from infected individuals show tremendous genetic and biologic diversity. To delineate the genetic determinants underlying specific biologic characteristics, such as rate of replication, cytopathic effects, and ability to infect macrophages and T4 lymphoid cells, generation of hybrid HIV using viruses which exhibit distinct biologic features is essential. To develop methods for generating hybrid HIV, we constructed truncated HIV proviral DNA plasmids. Upon digestion with restriction enzymes, these plasmid DNAs were cotransfected into human rhabdomyosarcoma cells to generate hybrid HIV. The hybrid HIVs derived by this method were infectious upon transmission to both phytohemagglutinin-stimulated peripheral blood lymphocytes and established human leukemic T-cell lines. The virus derived from molecular clone pHXB2 (HIVHTLV-III) productively infected CEMx174 cells. On the other hand, molecular clone pARV (HIVSF2)-derived virus did not show productive infection of CEMx174 cells when used as a cell-free virus. The hybrid HIV containing the 3' end of the genome from pARV and the 5' end of the genome from pHXB2 was effective in infecting CEMx174 cells, but the converse hybrid containing 5' pARV and 3' pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef play a role in the ability of the virus to infect a certain cell type. The intracellular ligation method should be useful in the analysis of related and unrelated HIV-1 isolates with common restriction enzyme cleavage sites.

The long terminal repeats (LTRs) of human immunodeficiency virus type 1 (HIV-1) strains from the central nervous systems of four patients with AIDS and of an HIV-1 isolate which is highly macrophage-tropic were isolated by using the polymerase chain reaction. In transient transfection assays, these LTRs demonstrated no significant difference in basal or stimulated levels of transcription in any of a variety of cell lines tested, compared with expression directed from the LTR of a T-lymphocyte-tropic strain of HIV-1. Chimeric viruses were created with the LTRs of the macrophage-tropic and brain-derived viruses ligated to the viral backbone from a T-lymphocyte-tropic strain. No change in cellular tropism was demonstrated with these chimeric viruses. Thus, unlike the LTRs of some murine retroviruses, the LTR of HIV-1 does not appear to play a major role in determining cellular tropism.

To establish the phenotype of human T-cell leukemia virus type 1 (HTLV-1)-infected cells in peripheral blood, the polymerase chain reaction was used to detect and quantitate viral DNA in subpopulations of leukocytes obtained from patients with tropical spastic paraparesis and asymptomatic carriers. HTLV-1 could not be detected in peripheral blood mononuclear cells thoroughly depleted of T lymphocytes (E- CD3-), nor could it be detected in highly enriched populations of B lymphocytes (E- CD19+), monocytes (E- CD14+), or natural killer cells (E- CD16+). T lymphocytes were strongly positive for HTLV-1, and fractionation of this population revealed that 90 to 99% of the HTLV-1 DNA segregated with the CD4+ CD8- and CD45RO+ subsets. No difference between the cell type distribution of HTLV-1 in the asymptomatic carrier and the subjects with tropical spastic paraparesis was evident. Southern hybridization of genomic DNA prepared from the peripheral blood of HTLV-1 carriers indicated that up to 10% of circulating leukocytes may carry the HTLV-1 provirus.

To evaluate the basis of the slow growth of many human immunodeficiency virus strains in monocytes/macrophages, various stages of the virus life cycle have been studied for their possible contribution to viral tropism. Although we found that monocytic U937 cells had a higher percentage of CD4-positive cells than T-lymphoid H9 cells, the human immunodeficiency virus strain grew much less efficiently in the monocytic line. Viral tropism was primarily determined during the early stages of the virus cycle, that is, sometime between binding of the virus to the cell surface and reverse transcription of viral genomic RNA. Once the virus entered the host cell, reverse transcription, use of the long terminal repeat, RNA expression, and production of virus particles was about as efficient in monocytes as in T cells. Thus, during viral entry into the host cell cytoplasm there is a major limiting event that is particularly inefficient in U937 cells and possibly in all monocytes/macrophages.

Simian acquired immunodeficiency syndrome is a fatal immunosuppressive disease caused by type D retroviruses such as simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1). The disease is characterized by generalized lymphadenopathy, opportunistic infections, and lymphoid depletion with defects in both humoral and cell-mediated immunity. To understand how SRV-1 infection relates to the immune defect, we studied in vivo-infected lymphocytes from SRV-1-positive macaques with and without clinical signs of immunosuppressive disease. B and T helper/inducer and T suppressor/cytotoxic lymphocytes were purified by panning or by flow cytometry. Neutrophils were purified by dextran sedimentation, and platelets were purified by low-speed centrifugation. In vitro infection studies were also done with HUT78, H9, K562, rhesus lung fibroblast, rhesus monkey kidney, and bat lung cells. SRV-1 in lymphocytes or culture supernatants was detected by the induction of syncytia in cocultivated Raji cells and was confirmed by immunofluorescence, electron microscopy, or reverse transcriptase assay. We found that B and T helper/inducer lymphocytes were infected in all animals tested. The number of infected T suppressor/cytotoxic cells was generally lower than that of the other cell subsets, and not all animals in this subset had SRV-1 infections. All other cells exposed in vitro to SRV-1, except bat lung cells, were able to be infected. These findings show that SRV-1 has a broad cell tropism for lymphoid and nonlymphoid cell types.