The major cellular sterol regulatory pathway is required for Andes virus infection.

The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.

Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies.

Puumala (PUUV) and Hantaan (HTNV) viruses are hantaviruses within the family Bunyaviridae and associated with Hemorrhagic Fever with Renal Syndrome (HFRS) in humans. Little is known about how these viruses interact with host cells, though pathogenic hantaviruses interact with α(v)ß(3) integrin. To study host cell interactions and rapidly test the ability of antibodies to prevent infection, we produced HTNV and PUUV pseudovirions on a vesicular stomatitis virus (VSV) core. Similar to replication-competent hantaviruses, infection was low-pH-dependent. Despite broad cell tropism, several human T cell lines were poorly permissive to hantavirus pseudovirions, compared to VSV, indicating a relative block to infection at the level of entry. Stable expression of α(v)ß(3) integrin in SupT1 cells did not restore infectivity. Finally, the pseudovirion system provided a rapid, quantitative, and specific method to screen for neutralizing antibodies in immune sera.

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

Expression of murine APOBEC3 alleles in different mouse strains and their effect on mouse mammary tumor virus infection.

Recent work has shown that mouse APOBEC3 restricts infection by mouse mammary tumor virus (MMTV) and murine leukemia virus (MLV) and that there are polymorphic APOBEC3 alleles found in different inbred mouse strains. For example, C57BL/6 mice, which are resistant to Friend MLV (F-MLV), encode a APOBEC3 gene different from that encoded by F-MLV-susceptible BALB/c mice; the predominant RNA produced in C57BL/6 mice lacks exon 5 (mA3(-5)) and encodes a protein with 15 polymorphic amino acids. It has also been reported that BALB/c mice produce only a variant RNA that lacks exon 2 (mA3(-2)). In this study, we examined the effect of these polymorphic APOBEC3 proteins on MMTV infection. We found that the major RNA made in C57BL/6 and B10.BR mice lacks exon 5 but that BALB/c and C3H/HeN mice predominantly express an RNA that contains all nine exons. In addition to producing the splice variant, C57BL/6 and B10.BR cells and tissues had levels of mA3 RNA fivefold higher than those from BALB/c and C3H/HeN mice. A cloned C57BL/6-derived mA3 protein lacking exon 5 inhibited MMTV infection better than a cloned full-length protein derived from 129/Ola RNA when packaged into MMTV virions. We also tested dendritic cells derived from different inbred mouse strains for their abilities to be infected by MMTV and showed that susceptibility to infection correlated with the presence of the exon 5-encoding allele. In vivo susceptibility to infection cosegregated with the inherited mA3 allele in a C57BL/6 x BALB/c backcross analysis. Moreover, virus produced in vivo in the mammary tissue of mA3 knockout and BALB/c mice was more infectious than that produced in the tissue of C57BL/6 mice. These data indicate that mA3 plays a role in the genetics of susceptibility and resistance to MMTV infection.

Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation.

Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin-positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)-positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin-positive, and TH-positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin-positive neural precursors.